Super fast-acting insulin compositions

ABSTRACT

Provided are methods for control of post-prandial glucose in diabetic subjects by administering a fast-acting insulin analog and a hyaluronidase degrading enzyme. The fast-acting insulin analog and a hyaluronidase are administered between 15 minutes before the meal and 30 minutes after commencing the meal.

RELATED APPLICATIONS

This application is a continuation of pending U.S. application. Ser. No.12/387,225, entitled “SUPER FAST-ACTING INSULIN COMPOSITIONS,” filedApr. 28, 2009, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/125,835, to Gregory Frost, IgorBilinsky, Daniel Vaughn and Barry Sugarman, entitled “SUPER FAST-ACTINGINSULIN COMPOSITIONS,” filed Apr. 28, 2008, and to U.S. ProvisionalApplication Ser. No. 61/127,044, to Gregory Frost, Igor Bilinsky, DanielVaughn and Barry Sugarman, entitled “SUPER FAST-ACTING INSULINCOMPOSITIONS,” filed May 9, 2008.

This application is related to corresponding International ApplicationNo. PCT/US2009/002625 to Gregory Frost, Igor Bilinsky, Daniel Vaughn andBarry Sugarman, entitled “SUPER FAST-ACTING INSULIN COMPOSITIONS,” whichalso claims priority to U.S. Provisional Application Ser. Nos.61/125,835 and 61/127,044.

The subject matter of each of the above-referenced applications isincorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS

An electronic version on compact disc (CD-R) of the Sequence Listing isfiled herewith in duplicate (labeled Copy #1 and Copy #2), the contentsof which are incorporated by reference in their entirety. Thecomputer-readable file on each of the aforementioned compact discs,created on May 15, 2012 is identical, 860 kilobytes in size, and titled3063Bseq.001.txt.

FIELD OF THE INVENTION

Provided are combinations, compositions and kits containing afast-acting insulin composition and a hyaluronan degrading enzymecomposition formulated for parenteral administration. Such products canbe used in methods of treating insulin-treatable diseases or conditions.Also provided are methods for administration of insulin and a hyaluronandegrading enzyme.

BACKGROUND

Diabetes results in chronic hyperglycemia due to the inability of thepancreas to produce adequate amounts of insulin or due to the inabilityof cells to synthesize and release the insulin appropriately.Hyperglycemia also can be experienced by critically ill patients,resulting in increased mortality and morbidity. Insulin has beenadministered as a therapeutic to treat patients having diabetes,including, for example, type 1 diabetes, type 2 diabetes and gestationaldiabetes, in order to mimic the endogenous insulin response that occursin normal individuals. Insulin also has been administered to criticallyill patients with hyperglycemia to control blood glucose levels.

Typically, fast-acting insulins are administered to such subjects inresponse to hyperglycemia or in anticipation of hyperglycemia, such asfollowing consumption of a meal, which can result in glycemic control.However, current fast-acting forms of insulins have a delay inabsorption and action, and therefore do not approximate the rapidendogenous insulin action. Thus, such formulations do not act quicklyenough to shut off hepatic glucose production that occurs shortly afterthis first phase of insulin release. Due to the delay in pharmacologicalaction, the fast-acting insulin preparations should be administered inadvance of meals in order to achieve the desired glycemic control.Further, the doses that must be administered lead to an extendedduration of action that contributes to hypoglycemia, and in many cases,obesity. Hence, there is a need for alternative insulin compositionsthat more effectively mimic the endogenous insulin response whenadministered to a subject, leading to more effective glycemic controland a reduction in the negative side-effects of insulin therapy, such asweight gain.

SUMMARY

Provided are super fast-acting insulin compositions that can act morerapidly and/or increase systemic exposure during a preselected timeperiod compared to fast-acting compositions. Hence, provided are superfast-acting insulin compositions. The compositions contain atherapeutically effective amount of a fast-acting insulin and an amountof a hyaluronan degrading enzyme to render the composition superfast-acting. The compositions are formulated for parenteraladministration, such as subcutaneous, intradermal or intramuscularadministration. Insulin dosage (amount administered) can be determinedby the quantity sufficient to achieve glycemic control, which can bedetermined empirically, such as by glucose challenge. Typically, a goalin treatment is to administer the lowest possible amount of insulin toachieve glycemic control and reduce the number of hyperglycemic and/orhypoglycemic events. The lower doses of insulin used in the superfast-acting insulin compositions can reduce the risk of weight gain andobesity in diabetic subjects. The compositions can be provided in anysuitable container or vehicle, such as in a sterile vial, syringe,cartridge, insulin pen, insulin pump or in a closed loop systemreservoir.

Provided herein are super fast-acting insulin compositions containing atherapeutically effective amount of a fast-acting insulin to controlblood glucose levels and an amount of a hyaluronan degrading enzymesufficient to render the composition a super fast-acting insulincomposition. Also provided are methods for making super fast-actinginsulin compositions, such as any super fast-acting insulin compositionsdescribed herein, by selecting a fast-acting insulin and combining itwith a sufficient amount of hyaluronan degrading enzyme to render thecomposition a super fast-acting insulin composition. In some examples ofthe compositions and methods of making the compositions, thetherapeutically effective amount of fast-acting insulin is from or fromabout 10 U/mL to or to about 500 U/ml insulin, and the sufficient amountof a hyaluronan degrading enzyme to render the composition a superfast-acting insulin composition is functionally equivalent to at leastor about 1 U/mL, 2 U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL,9 U/mL, 10 U/mL, 15 U/mL, 20 U/mL or 25 U hyaluronidase activity/mL. Insome examples, the sufficient amount of a hyaluronan degrading enzyme torender the composition a super fast-acting insulin composition isfunctionally equivalent to at least or about 30 or 35 Unitshyaluronidase activity/mL. For example, the amount of fast-actinginsulin in the compositions can be or be about 10 U/mL, 20 U/mL, 30U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, 100 U/mL,150 U/mL, 200 U/mL, 250 U/mL, 300 U/mL, 350 U/mL, 400 U/mL, 450 U/ml or500 U/mL, and the amount of hyaluronan degrading enzyme in thecompositions can be functionally equivalent to or to about 1 U/mL, 2U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL,15 U/mL, 20 U/mL, 25 U/mL, 30 U/mL, 35 U/mL, 37.5 U/mL, 40 U/mL, 50U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, 100 U/mL, 200 U/mL, 300 U/mL,400 U/mL, 500 U/mL, 600 U/mL, 700 U/mL, 800 U/mL, 900 U/mL, 1000 U/ml,2000 U/mL, 3000 U/mL or 5000 U/mL. The volume of the composition can be,for example, at or about 1 mL, 3 mL, 5 mL, 10 mL, 20 mL or 50 mL. Insome examples, the composition is formulated for delivery by a closedloop system, an insulin pen or an insulin pump, and can be formulatedfor single dose administration or multiple dose administration.

In some embodiments, the therapeutically effective amount of thefast-acting insulin is less than the therapeutically effective amount offast-acting insulin required to achieve the same therapeutic effect inthe absence of the hyaluronan degrading enzyme. The amount of hyaluronandegrading enzyme is sufficient to achieve a systemic exposure to insulinthat is at least or about 30% greater over the first 3, 6, 9, 12, 15,20, 25, 30, 35, 40, 50 or 60 minutes following parenteral administrationthan the systemic exposure over the same time period followingparenteral administration of the same fast-acting insulin without ahyaluronan degrading enzyme and/or is sufficient to achieve systemicglucose metabolism (sometimes referred to herein as glucose clearance)that is at least or about 30% greater over the first 30, 45, 60, 90, 120or 180 minutes following administration than the systemic glucosemetabolism over the same period following parenteral administration ofthe same fast-acting insulin without a hyaluronan degrading enzyme. Inall compositions provided herein and methods provided herein, theamounts of each component can vary depending upon the subject to whomthe compositions is administered and/or the particular fast-actinginsulin (or mixture thereof) that is provided. If necessary, the amountscan be determined empirically.

Provided are insulin compositions that contain a therapeuticallyeffective amount of a fast-acting insulin and an amount of a hyaluronandegrading enzyme. The amount of hyaluronan degrading enzyme issufficient to achieve a systemic exposure to insulin that is at least orabout 30% greater over the first 30 to 40 minutes followingadministration than the systemic exposure over the same period followingparenteral administration of the same fast-acting insulin in the absenceof the hyaluronan degrading enzyme.

The amount of hyaluronan degrading enzyme can be sufficient so that theresulting super fast-acting insulin composition results in a bloodglucose level increase after the first 30, 45, 60, 90, 120 or 180minutes following parenteral administration that is at least or about20% to 30% lower than the increase in blood glucose levels over the sametime period following parenteral administration of the same fast-actinginsulin without a hyaluronan degrading enzyme. The increase in bloodglucose level can be at least or about 30%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75% or 80% less than the increase in blood glucoselevel following parenteral administration of the fast-acting insulinwithout a hyaluronan degrading enzyme.

Also provided are super fast-acting insulin compositions that contain atherapeutically effective amount of a fast-acting insulin and an amountof hyaluronan degrading enzyme that is sufficient to achieve systemicglucose metabolism that is at least or about 30% greater than systemicglucose clearance (i.e. metabolism) over the first 60 minutes followingparenteral administration of the same fast-acting insulin without ahyaluronan degrading enzyme.

In the super fast-acting insulin compositions provided herein, exemplaryamounts of insulin (i.e., the amount that the composition provides for asingle dosage) are at or about 0.05 Units, 0.06 Units, 0.07 Units, 0.08Units, 0.09 Units, 0.1 Units, 0.2 Units, 0.3 Units, 0.4 Units, 0.5Units, 0.6 Units, 0.7 Units, 0.8 Units, 0.9 Units, 1 Unit, 2 Units, 5Units, 10 Units, 15 Units, 20 Units, 25 Units, 30 Units, 35 Units, 40Units, 50 Units or 100 Units. Exemplary amounts of hyaluronan degradingenzyme include an amount functionally equivalent to at or about 0.3Units, 0.5 Units, 1 Unit, 3 Units, 5 Units, 10 Units, 20 Units, 30Units, 40 Units, 50 Units, 100 Units, 150 Units, 200 Units, 250 Units,300 Units, 350 Units, 400 Units, 450 Units, 500 Units, 600 Units, 700Units, 800 Units, 900 Units, 1000 Units, 2,000 Units, 3,000 Units, 4,000or more of hyaluronidase activity.

The super fast-acting insulin compositions provided herein can achieveprandial (e.g. 0-4 hours post administration) systemic exposure toinsulin that is at least or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, 300% or 400% greater thanthe systemic exposure following parental administration of insulin inthe absence of the hyaluronan degrading enzyme. The super fast-actinginsulin compositions provided herein can achieve systemic glucosemetabolism (i.e., a quantification of the removal of glucose from bloodexpressed either as a rate (amount/time) or the total amount during apredetermined period of time) that is at least or about 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, 250%, 300%, 350%or 400% greater than the metabolism of blood glucose followingparenteral administration of insulin without a hyaluronan degradingenzyme.

The super fast-acting insulin compositions provided herein optionallyinclude a chelating agent, such as, but not limited toethylenediaminetetraacetic acid (EDTA) or ethylenediaminetetraacetate.The chelating agent can be provided as a complex with a metal at orabout equimolar concentrations therewith, such as the chelating agentcomplex calcium EDTA. The concentration of calcium EDTA is or is about0.02 mM, 0.04 mM, 0.06 mM, 0.08 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 5 mM, 10 mM, 15 mM or 20 mM.

The super fast-acting insulin compositions herein generally includezinc. The concentration of zinc typically is or is about 0.002milligrams per 100 units of insulin (mg/100 U), 0.005 mg/100 U, 0.01mg/100 U, 0.012 mg/100 U, 0.014 mg/100 U, 0.016 mg/100 U, 0.017 mg/100U, 0.018 mg/100 U, 0.02 mg/100 U, 0.022 mg/100 U, 0.024 mg/100 U, 0.026mg/100 U, 0.28 mg/100 U, 0.03 mg/100 U, 0.04 mg/100 U, 0.05 mg/100 U,0.06 mg/100 U, 0.07 mg/100 U, 0.08 mg/100 U or 0.1 mg/100 U. In general,fast-acting insulins are formulated with zinc; the amount used hereincan be an amount that retains the same concentration of zinc whencombined with the hyaluronan degrading enzyme. Exemplary compositionscan contain calcium EDTA and zinc at molar ratios of or about 0.5:1,1:1, 1.5:1, 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1,90:1, 100:1, 300:1 or 1000:1, such as about or 0.010-0.50 mg zinc, suchas 0.017 mg zinc per 100 U human insulin, and 0.1 to 50 mM calcium EDTA.Other exemplary super fast-acting insulin compositions contain zinc in amolar ratio of about 1:3 to the fast-acting insulin and calcium EDTA ata molar ratio of about 1:3 to 10:1 to the fast-acting insulin.

The super fast-acting insulin compositions also optionally include atonicity modifier, such as, but not limited to, an amino acid,polyalcohol, such as glycerol, and/or a salt, such as, sodium chloride.The osmolarity of the composition can be or is about 200 mOsm/kg, 220mOsm/kg, 240 mOsm/kg, 260 mOsm/kg, 280 mOsm/kg, 300 mOsm/kg, 320mOsm/kg, 340 mOsm/kg, 360 mOsm/kg, 380 mOsm/kg or 400 mOsm/kg. The pH issuitable for parenteral administration, such as about or 5.5 to 8.5,particularly, 6 to 8, such as, about or is 6, 6.2, 6.4, 6.6, 6.8, 7,7.2, 7.4, 7.6, 7.8 or 8. The compositions can optionally include astabilizer for the fast-acting insulin, a stabilizer for the hyaluronandegrading enzyme or both. Stabilizers include, but are not limited to, adetergent, a polyalcohol, a metal, a salt, a cosolvent and/or a protein.Exemplary of such stabilizers is serum albumin and/or polysorbate, at aconcentration sufficient to achieve greater stability of the compositionand/or a component. Serum albumin can be included at a concentration ofor about 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL or 1 mg/mL. Polysorbate can beincluded, for example, at a concentration of or about 0.001%, 0.002%,0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%,0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%. Other optionalingredients include, for example, an oxygen scavenger, such as ascorbicacid, ascorbate, citric acid, citrate, methionine, which can be at aconcentration of 1 mM, 2 mM, 3 mM, 5 mM, 10 mM, or 20 mM, and/or albuminand/or a preservative, such as a compound that contains an aromaticring, for example, m-cresol or phenol.

The fast-acting insulin can be, for example, monomeric or multimeric,such as dimeric or hexameric. Among the fast-acting insulins are regularinsulins, such as, but not limited to, human insulin or pig insulin,such as an insulin with an A chain containing or having a sequence ofamino acids set forth in SEQ ID NO:103, and a B chain containing orhaving a sequence of amino acids set forth in SEQ ID NO:104, or aninsulin with an A chain containing or having a sequence of amino acidsset forth as amino acid residue positions 88-108 of SEQ ID NO:123 and aB chain containing or having a sequence of amino acids set forth asamino acid residue positions 25-54 of SEQ ID NO:123. The insulin can bea recombinant insulin or can be synthesized or partially-synthesized orcan be isolated from a natural source. The insulin can be an insulinanalog. Exemplary of insulin analogs is an insulin analog selected fromamong an insulin with an A chain containing or having a sequence ofamino acids set forth in SEQ ID NO:103 and a B chain containing orhaving a sequence of amino acids set forth in any of SEQ ID NOS:147-149.In some exemplary super fast-acting insulin compositions, thefast-acting insulin is a fast-acting human insulin. Further, the superfast-acting insulin compositions can contain mixtures of insulins. Themixtures can be fast-acting insulins, or mixtures of a fast-actinginsulin and also a slower-acting insulin(s), such as a basal-actinginsulin.

Hyaluronan degrading enzymes contained in the compositions andcombinations provided herein include, for example, hyaluronidases, suchas animal, including human, hyaluronidases, particularly soluble formsthereof. Exemplary hyaluronan degrading enzymes are hyaluronidases,particularly soluble hyaluronidases, such as a PH20, or a truncated formthereof. The PH20 can be, for example, an ovine, bovine or truncatedhuman PH20. Included are those that contain or have a sequence of aminoacids set forth in any of SEQ ID NOS:1-39 and 67-96 and truncated formsthereof or allelic variants, species variants or other variants thereof.Truncated human PH20, particularly soluble truncated forms, includes anyfrom among polypeptides having a sequence of amino acids set forth inany of SEQ ID NOS:4-9, or allelic variants and other variants thereof.Variants of the hyaluronidases typically have at least 40%, 50%, 60%,70%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity with any of SEQ ID NOS: 1-39 and 67-96,particularly with soluble forms, and retain hyaluronidase activity. Thesoluble hyaluronidase can be the composition that is rHuPH20.

The hyaluronan degrading enzyme can be a chondroitinase, such as, butnot limited to, chondroitin ABC lyase, chondroitin AC lyase andchondroitin C lyase. Exemplary chondroitinases have or contain asequence of amino acids set forth in any of SEQ ID NOS:98-100, ortruncated forms thereof or allelic variants, species variants and othervariants thereof. Variants typically have at least 40%, 50%, 60%, 70%,80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity with a polypeptide set forth in any of SEQ IDNOS. 98-100 or with a wild-type chondroitinase.

The super fast-acting insulin compositions provided herein can beformulated for multiple dosage administration, for dilution to a desireddose or for single dose administration. Exemplary therapeuticallyeffective amounts of insulin depend upon the insulin in the compositionand the subject to whom the composition is administered. Such singledosage amounts include, for example, at or about 0.05 Units, 0.06 Units,0.07 Units, 0.08 Units, 0.09 Units, 0.1 Units, 0.2 Units, 0.3 Units, 0.4Units, 0.5 Units, 0.6 Units, 0.7 Units, 0.8 Units, 0.9 Units, 1 Unit, 2Units, 5 Units, 10 Units, 15 Units, 20 Units, 25 Units, 30 Units, 35Units, 40 Units, 50 Units or 100 Units. In such compositions, the amountof hyaluronan degrading enzyme can be or is functionally equivalent toat or about 0.3 Units, 0.5 Units, 1 Unit, 2 Units, 3 Units, 4 Units, 5Units, 10 Units, 20 Units, 30 Units, 40 Units, 50 Units, 100 Units, 150Units, 200 Units, 250 Units, 300 Units, 350 Units, 400 Units, 450 Units,500 Units, 600 Units, 700 Units, 800 Units, 900 Units or 1000 Units ofhyaluronidase activity.

The super fast-acting insulin compositions can be formulated fordelivery by a pump. Provided are closed loop systems for controllingblood glucose levels. The systems are any known to those of skill in theart, but modified by containing the fast-acting insulin and hyaluronandegrading enzyme as described herein and suitable dosing or programmingto deliver therapeutic dosages of fast-acting insulin and a hyaluronandegrading enzyme to produce a super fast-acting insulin composition. Theclosed loop systems can include a reservoir containing a fast-actinginsulin and a hyaluronan degrading enzyme, where the hyaluronandegrading enzyme is present in an amount sufficient to render theresulting combination a super fast-acting insulin composition. Inanother embodiment a closed loop system for controlling blood glucoselevels is provided that contains a reservoir containing a fast-actinginsulin and a second reservoir containing a hyaluronan degrading enzyme.

The closed loop systems optionally can include one or more of a glucosesensor, a delivery system to deliver the hyaluronan degrading enzyme andfast-acting insulin and software programmed to integrate the pumping andmonitoring functions, whereby hyaluronan degrading enzyme andfast-acting insulin are delivered to achieve glycemic control thatmimics the glycemic control in a non-diabetic subject. The closed loopsystems also can contain in a separate reservoir or mixed with thefast-acting insulin and/or hyaluronan, a slower-acting, such as a basal,insulin. The system also can include any of the optional ingredientsnoted above. The fast-acting insulin and hyaluronan degrading enzyme caninclude any of those described above.

In the closed loop system, the reservoir containing the fast-actinginsulin can contain a sufficient number of units to maintain glycemiccontrol for at least half of a day, one day or more and can contain ator about 0.1 Units, 0.2 Units, 0.3 Units, 0.4 Units, 0.5 Units, 0.6Units, 0.7 Units, 0.8 Units, 0.9 Units, 1 Unit, 2 Units, 5 Units, 10Units, 15 Units, 20 Units, 25 Units, 30 Units, 35 Units, 40 Units, 50Units, 100 Units, 200 Units, 300 Units, 400 Units, 500 Units, 600 Units,700 Units, 800 Units, 900 Units, 1000 Units, 2000 Units, 5000 Units,6000 Units, 7000 Units or more of insulin. The closed loop system candeliver any desired amounts or dose increments of insulin and/orhyaluronan degrading enzyme, such as at or about 0.05 Units, 0.1 Units,0.2 Units, 0.3 Units, 0.4 Units, 0.5 Units, 0.6 Units, 0.7 Units, 0.8Units, 0.9 Units, 1 Unit, 2 Units, 5 Units, 10 Units, 15 Units, 20Units, 25 Units, 30 Units, 35 Units, 40 Units, 50 Units or more ofinsulin per increment. The reservoir containing the hyaluronan degradingenzyme can contain an amount of hyaluronan degrading enzyme that isfunctionally equivalent to at or about 1 Unit, 5 Units, 10 Units, 20Units, 30 Units, 40 Units, 50 Units, 100 Units, 150 Units, 200 Units,250 Units, 300 Units, 350 Units, 400 Units, 450 Units, 500 Units, 600Units, 700 Units, 800 Units, 900 Units, 1000 Units, 2,000 Units, 3,000Units, 4,000 Units, 5000 Units, 6,000 Units, 7,000 Units, 8,000 Units,9,000 Units, 10,000 Units, 20,000 Units or more hyaluronidase activity,and can deliver the hyaluronan degrading enzyme in individual doseincrements of an amount of hyaluronan degrading enzyme that isfunctionally equivalent to at or about, for example, 0.3 Units. 0.5Units, 1 Unit, 2 Units, 3 Units, 5 Units, 10 Units, 20 Units, 30 Units,40 Units, 50 Units, 100 Units, 150 Units or more of hyaluronidaseactivity.

Also provided are combinations containing a first composition containingfrom or from about 10 U to or to about 500 U insulin, and a secondcomposition containing sufficient amount of hyaluronan degrading enzymethat, when administered with the insulin, renders the fast-actinginsulin a superfast acting insulin. The sufficient amount of hyaluronandegrading enzyme is functionally equivalent to least or about 1 U/mL, 2U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL,15 U/mL, 20 U/mL or 25 U hyaluronidase activity/mL. In some examples,the sufficient amount of hyaluronan degrading enzyme is functionallyequivalent to at least or about 35 U hyaluronidase activity/mL. Forexample, the amount of hyaluronan degrading enzyme in the secondcomposition can be functionally equivalent to or to about 1 U/mL, 2U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL,15 U/mL, 20 U/mL, 25 U/mL, 30 U/mL, 35 U/mL, 37.5 U/mL, 40 U/mL, 50U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, 100 U/mL, 200 U/mL, 300 U/mL,400 U/mL, 500 U/mL, 600 U/mL, 700 U/mL, 800 U/mL, 900 U/mL, 1000 U/ml,2000 U/mL, 3000 U/mL or 5000 U/mL. In some examples, the amount offast-acting insulin in the first composition is or is about 10 U/mL, 20U/mL, 30 U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, 100U/mL, 150 U/mL, 200 U/mL, 250 U/mL, 300 U/mL, 350 U/mL, 400 U/mL, 450U/ml or 500 U/mL.

Also provided are combinations of a first composition containing ahyaluronan degrading enzyme and a second composition containing afast-acting insulin. The compositions are formulated for parenteraladministration. In some instances, the amount of hyaluronan degradingenzyme is sufficient if mixed with the second composition to render theresulting composition a super fast-acting insulin composition. In otherinstances, the amount of the hyaluronan degrading enzyme is sufficientif administered prior to the administration of the first composition torender the fast-acting insulin composition a super fast-acting insulincomposition.

In the combinations provided herein, the fast-acting insulins andhyaluronan degrading enzymes and other components are as described abovefor the compositions. Kits containing the combinations also areprovided. The composition of insulin can be formulated to administer aprandial dosage for a single meal, such as, but not limited to, about0.001 U/kg, 0.005 U/kg, 0.01 U/kg, 0.02 U/kg, 0.03 U/kg, 0.04 U/kg, 0.05U/kg, 0.06 U/kg, 0.07 U/kg, 0.08 U/kg, 0.09 U/kg, 0.10 U/kg, 0.11 U/kg,0.12 U/kg, 0.13 U/kg, 0.14 U/kg, 0.15 U/kg, 0.20 U/kg, 0.25 U/kg, 0.30U/kg, 0.40 U/kg, 0.50 U/kg, 1 U/kg, 1.5 U/kg, or 2 U/kg. The amount ofhyaluronan degrading enzyme is formulated to administer to the subject aprandial dosage for a single meal and, for example, is or is about 0.3U, 0.5 U, 1 U, 2 U, 3 U, 4 U, 5 U, 10 U, 20 U, 30 U, 40 U, 50 U, 100 U,150 U, 200 U, 250 U, 300 U, 350 U, 400 U, 450 U, 500 U, 600 U, 700 U,800 U, 900 U, 1000 U, 2,000 U, 3,000 U, 4,000 U, 5,000 U or more. Thecompositions in the combination can be formulated for subcutaneousadministration.

Provided are methods in which the super fast-acting insulin compositionsand combinations provided herein are administered. Typically suchadministration is parenteral administration, such as intravenous,subcutaneous or via any suitable route. In any of the methods providedherein, the fast-acting insulin and hyaluronan degrading enzyme can beadministered separately, intermittently, or together in separatecompositions or co-formulated. Also provided are methods for controllingglucose levels in a subject by administering any of the superfast-acting insulin compositions or combinations provided herein. Insome instances, the compositions or combinations are administered as aprandial dosage, including such as administered less than or about 20,10, 5 minutes prior to a meal, to less than or about 10 minutes after ameal or with the meal.

Also provided are methods that involve instructing a patient toadminister a fast-acting insulin composition less than or about 20, 10,5 minutes prior to a meal, to less than or about 30 minutes after ameal, wherein the fast-acting insulin is co-administered with asufficient amount of hyaluronan-degrading enzyme to render thefast-acting insulin composition a super fast acting composition. Thefast-acting insulin and hyaluronan degrading enzyme can be co-formulatedor provided separately for co-administration. In such methods, thepatient can be instructed to administer the fast-acting insulincomposition at or at about the time of ingestion of a meal. In someexamples, the instructions are written. In other examples, theinstructions are oral.

Provided are methods for controlling blood glucose levels in a subject,by administering to a subject a hyaluronan degrading enzyme and afast-acting insulin, where the hyaluronan degrading enzyme andfast-acting insulin are administered in sufficient amounts to

a) obtain a maximal increase in insulin concentration in the blood thatis at least or about 20% to 30% greater than the maximal increase ininsulin concentration in the blood obtained after administration of thefast-acting insulin in the same manner in the absence of a hyaluronandegrading enzyme; and/or

b) reduce the amount of time taken to reach the maximal insulinconcentration in the blood to not more than 80% of the time taken toreach the maximal insulin concentration in the blood when thefast-acting insulin is administered in the same manner in the absence ofa hyaluronan degrading enzyme; and/or

c) increase the insulin concentration 15 minutes after administration byat least or about 50, 60, 70, 80, 90 or 100 pmol/L.

By virtue of the methods, maximal increase in insulin concentration inthe blood is at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 120%, 140%, 160%, 180%, 200%, 250%, 300%, 350% or 400% greaterthan the maximal increase in insulin concentration in the absence of thehyaluronan degrading enzyme. The time taken to reach the maximal insulinconcentration in the blood can be reduced no more than 80% of the timetaken to reach the maximal insulin concentration in the blood in theabsence of the hyaluronan degrading enzyme. For example, the insulinconcentration following administration of a 20 U dose of insulin 15minutes after administration can be increased by at least or about 60pmol/L, 80 pmol/L, 100 pmol/L, 120 pmol/L, 140 pmol/L, 160 pmol/L, 180pmol/L, or 200 pmol/L.

In exemplary embodiments, the diabetic subjects have either Type 1 orType 2 diabetes, and the amount of fast-acting insulin administered tothe subject is reduced compared to when the fast-acting insulin isadministered in the same manner in the absence of a hyaluronan degradingenzyme. For example, the amount of fast-acting insulin administered to aType 1 diabetic subject can be reduced by at least or about 5%, 10%,20%, 30%, 40%, 50%, 60%, or more, and the amount of fast-acting insulinadministered to a Type 2 diabetic subject can be reduced by at least orabout 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more.

Also provided are methods for controlling or preventing weight gainand/or obesity in a diabetic subject, such as weight gain associatedwith prandial insulin therapy. Typically, this is achieved byadministering a hyaluronan degrading enzyme and a fast-acting insulin ata dose that is less than the dose of a fast-acting insulin whenadministered in the absence of a hyaluronan degrading enzyme. Thediabetic subjects can be obese or at risk of obesity, and can have Type1 diabetes, Type 2 diabetes, gestational diabetes or other diabetes.Exemplary of diabetic subjects are Type 2 diabetic subjects. In oneexample, controlling or preventing obesity in a diabetic subject isachieved by administering to an obese diabetic subject or a diabeticsubject at risk of obesity a therapeutically effective dosage of afast-acting insulin in combination with hyaluronan degrading enzyme. Thecomposition can be administered at or around mealtime, and

a) the amount of hyaluronan degrading enzyme is sufficient to render theadministered fast-acting insulin a super fast-acting insulin; and

b) the dosage of fast-acting insulin achieves substantially the samedegree of prandial glucose clearance within the first 40 minutesfollowing administration as a higher dosage of the same fast-actinginsulin administered in the same manner in the absence of the hyaluronandegrading enzyme. The dosage of the fast-acting insulin in the superfast-acting insulin composition, compared to the higher dose offast-acting insulin, has a reduced tendency to cause post-prandialhypoglycemia and obesity. In exemplary embodiments, the diabeticsubjects have Type 2 diabetes, and the amount of fast-acting insulinadministered to the subject is reduced compared to when the fast-actinginsulin is administered in the same manner in the absence of ahyaluronan degrading enzyme. For example, the amount of fast-actinginsulin administered to a Type 2 diabetic subject can be reduced by atleast or about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% ormore.

Also provided are methods for reducing or preventing weight gainassociated with prandial insulin therapy by subcutaneously administeringto a diabetic subject at risk for weight gain from prandial insulintherapy, at or around mealtime, an insulin composition containing afast-acting insulin and a hyaluronan degrading enzyme, such that theamount of fast-acting insulin administered to treat postprandialhyperglycemia in the subject is reduced compared to the amount of thesame fast-acting insulin required to treat the hyperglycemia whenadministered in the same manner in the absence of the hyaluronandegrading enzyme. The reduced amount of fast-acting insulin renders thecomposition containing the hyaluronan degrading enzyme less likely tocause weight gain in the subject. For example, a Type 2 diabetic subjectcan be administered an insulin composition as described above containingan amount of fast-acting insulin that is at least or about 5%, 10%, 15%,20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% less than the amount of thesame fast-acting insulin required to treat the hyperglycemia whenadministered in the same manner in the absence of the hyaluronandegrading enzyme. In some instances, the insulin composition isadministered in a chronic regimen of prandial insulin therapy.

Provided herein are methods for reducing or preventing weight gain in adiabetic subject by administering to a diabetic subject at risk ofweight gain from prandial insulin therapy, a course of subcutaneousprandial insulin therapy over a period of at least thirty days. Theprandial insulin dosages administered in the course of the therapycontain a combination of a fast-acting insulin and a hyaluronandegrading enzyme. The amount of hyaluronan degrading enzyme in eachdosage is sufficient to render the fast-acting insulin a superfast-acting insulin composition, and the amount of fast-acting insulincontained in the dosage to treat the subject's postprandialhyperglycemia is lower than an amount of the same fast-acting insulinrequired to treat the hyperglycemia in the absence of the hyaluronandegrading enzyme. Such a course of prandial insulin therapy can resultin less weight gain than a similar course of therapy using higherdosages of fast-acting insulin in the absence of hyaluronan degradingenzyme.

Also provided are methods for controlling glucose levels in a subject byadministering to the subject a prandial dosage of super fast-actinginsulin composition, where:

a) the super fast-acting insulin composition comprises a therapeuticallyeffective amount of a fast-acting insulin and a hyaluronan degradingenzyme;

b) the fast-acting insulin is a regular insulin;

c) the dosage is administered, or recommended for prandial orpreprandial administration, closer to mealtime than the same or agreater dosage of the same fast-acting regular insulin administered bythe same route of administration in the absence of a hyaluronandegrading enzyme; and

d) the dosage of the super fast-acting insulin composition has at leastthe same therapeutic effect as the fast-acting regular insulin withoutthe hyaluronan degrading enzyme.

The super fast-acting insulin composition for example, is administered,or recommended for administration, less than or about 20 minutes priorto a meal, to less than or about 10 or 20 minutes after a meal.Typically, the dosage of the fast-acting insulin in the superfast-acting insulin composition is less than or equal to the dosage ofthe fast-acting insulin administered by the same route in the absence ofthe hyaluronan degrading enzyme.

In practicing any of the methods provided herein, the compositions canbe administered via any suitable route and using any suitable device orcontainer, such as via syringe, insulin pen, insulin pump or closed loopsystem. The compositions or combinations can contain any of thefast-acting insulins and hyaluronan degrading enzymes described above,with any of the additional reagents as described above. The amountinsulin in the composition administered to the subject can be a prandialdosage for a single meal and is or is about 0.001 U/kg, 0.005 U/kg, 0.01U/kg, 0.02 U/kg, 0.05 U/kg to 0.30 U/kg, such as 0.05 U/kg, 0.06 U/kg,0.07 U/kg, 0.08 U/kg, 0.09 U/kg, 0.10 U/kg, 0.11 U/kg, 0.12 U/kg, 0.13U/kg, 0.14 U/kg, 0.15 U/kg, 0.20 U/kg, 0.25 U/kg, 0.30 U/kg, 0.40 U/kg,0.50 U/kg, 1.0 U/kg, 1.5/kg or 2 U/kg. The amount of hyaluronandegrading enzyme administered to the subject is for co-administration(separately, intermittently, or together in separate compositions orco-formulated) with a prandial dosage of fast-acting insulin for asingle meal. The amount of hyaluronan degrading enzyme can be or isabout 0.3 U, 0.5 U, 1 U, 2 U, 5 U, 10 U, 20 U, 30 U, 40 U, 50 U, 100 U,150 U, 200 U, 250 U, 300 U, 350 U, 400 U, 450 U, 500 U, 600 U, 700 U,800 U, 900 U, 1000 U, 2,000 U, 3,000 U, 4,000 Units, 5,000 U or more.

Provided are articles of manufacture containing packaging material andany of the super fast-acting insulin compositions or combinations withinthe packaging material with optional instructions for administration toa diabetic subject.

The fast-acting insulins, insulins, hyaluronan degrading enzymes andother components include those as described above. In particular, thecompositions and combinations provided herein are administered.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the pharmacokinetic profiles of the fast-acting insulinanalog, Humalog® insulin, and the fast-acting regular insulin, Humulin®R insulin, when administered subcutaneously with or withoutco-administration of rHuPH20. The plasma insulin concentration atvarious timepoints following administration to normal healthy subjectsusing a Hyperinsulinemic-Euglycemic Clamp procedure was determined byradioimmunoassay (RIA).

FIG. 2 depicts the pharmacodynamic profiles of the fast-acting insulinanalog, Humalog® insulin, and the fast-acting regular insulin, Humulin®R insulin, when administered subcutaneously with or withoutco-administration of rHuPH20 using a Hyperinsulinemic-Euglycemic Clampprocedure. The glucose infusion rate that was required to maintain bloodglucose levels between 90-110 mg/dL following insulin administration tonormal healthy subjects was determined.

DETAILED DESCRIPTION Outline

A. Definitions

B. “Super fast-acting” insulin

-   -   1. Overview of Insulin, Diabetes and Existing Fast-Acting        Insulin Therapies    -   2. Pharmacodynamics and Pharmacokinetics of a Super Fast-Acting

Insulin Composition

C. Insulin Polypeptides and Formulation

D. Hyaluronan degrading enzymes

-   -   1. Hyaluronidases        -   a. Mammalian-type hyaluronidases        -   b. Bacterial hyaluronidases        -   c. Hyaluronidases from leeches, other parasites and            crustaceans    -   2. Other hyaluronan degrading enzymes    -   3. Soluble hyaluronan degrading enzymes        -   a. Soluble Human PH20        -   b. Recombinant soluble Human PH20 (rHuPH20)    -   4. Glycosylation of hyaluronan degrading enzymes    -   5. Modifications of hyaluronan degrading enzymes to improve        their pharmacokinetic properties

E. Methods of Producing Nucleic Acids encoding a soluble Hyaluronidaseand Polypeptides Thereof

-   -   1. Vectors and Cells    -   2. Linker Moieties    -   3. Expression        -   a. Prokaryotic Cells        -   b. Yeast Cells        -   c. Insect Cells        -   d. Mammalian Cells        -   e. Plants    -   4. Purification Techniques

F. Preparation, Formulation and Administration of Insulin and SolubleHyaluronidase Polypeptides

-   -   1. Formulations        -   Lyophilized Powders    -   2. Dosage and Administration        -   Mode of Administration            -   a. Syringes            -   b. Insulin pen            -   c. Insulin pumps and other insulin delivery devices            -   d. Closed loop system

G. Methods of Assessing Activity, Bioavailability and Pharmacokinetics

-   -   1. Pharmacokinetics, pharmacodynamics and tolerability    -   2. Biological Activity        -   a. Insulin        -   b. Hyaluronan degrading enzymes

H. Therapeutic Uses

-   -   1. Diabetes Mellitus        -   a. Type 1 diabetes        -   b. Type 2 diabetes        -   c. Gestational diabetes    -   2. Insulin therapy for critically ill patients

I. Combination Therapies

J. Articles of Manufacture and Kits

K. Examples

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, Genbank sequences, databases,websites and other published materials referred to throughout the entiredisclosure herein, unless noted otherwise, are incorporated by referencein their entirety. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, “insulin” refers to a hormone, precursor or a syntheticor recombinant analog thereof that acts to increase glucose uptake andstorage and/or decrease endogenous glucose production. An exemplaryhuman insulin is translated as a 110 amino acid precursor polypeptide,preproinsulin (SEQ ID NO:101), containing a 24 amino acid signal peptidethat directs the protein to the endoplasmic reticulum (ER) wherein thesignal sequence is cleaved, resulting in proinsulin (SEQ ID NO:102).Proinsulin is processed further to release the 31 amino acid C- orconnecting chain peptide (corresponding to amino acid residues 57 to 87of the preproinsulin polypeptide set forth in SEQ ID NO:101, and toamino acid residues 33 to 63 of the proinsulin polypeptide set forth inSEQ ID NO:102). The resulting insulin contains a 21 amino acidA-chain.(corresponding to amino acid residues 90 to 110 of thepreproinsulin polypeptide set forth in SEQ ID NO:101, and to amino acidresidues 66 to 86 of the proinsulin polypeptide set forth in SEQ IDNO:102) and a 30 amino acid B-chain (corresponding to amino acidresidues 25 to 54 of the preproinsulin polypeptide set forth in SEQ IDNO:101, and to amino acid residues 1 to 30 of the proinsulin polypeptideset forth in SEQ ID NO:102) which are cross-linked by disulfide bonds. Aproperly cross-linked human insulin contains three disulfide bridges:one between position 7 of the A-chain and position 7 of the B-chain, asecond between position 20 of the A-chain and position 19 of theB-chain, and a third between positions 6 and 11 of the A-chain.Reference to insulin includes preproinsulin, proinsulin and insulinpolypeptides in single-chain or two-chain forms, truncated forms thereofthat have activity, and includes allelic variants and species variants,variants encoded by splice variants, and other variants, such as insulinanalogs, including polypeptides that have at least 40%, 45%, 50%, 55%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to the precursor polypeptide set forth in SEQ ID NO:101 or themature form thereof. Exemplary insulin analogs include those set forthin SEQ ID NOS:147-149, 152, and those containing an A-chain set forth inSEQ ID NOS:150, 156, 158, 160, 162 and 164 and/or a B chain set forth inSEQ ID NOS:151, 153-155, 157, 159, 161, 163 and 165.

Exemplary insulin polypeptides are those of mammalian, including human,origin. Exemplary amino acid sequences of insulin of human origin areset forth in SEQ ID NOS: 101-104. Exemplary insulin analogs includethose set forth in SEQ ID NOS:147-149, 152, and those containing anA-chain set forth in SEQ ID NOS:150, 156, 158, 160, 162 and 164 and/or aB chain set forth in SEQ ID NOS:151, 153-155, 157, 159, 161, 163 and165. Insulin polypeptides also include any of non-human originincluding, but not limited to, any of the precursor insulin polypeptidesset forth in SEQ ID NOS:105-146. Reference to an insulin includesmonomeric and multimeric insulins, including hexameric insulins, as wellas humanized insulins.

As used herein, “fast-acting insulin” refers to any insulin orfast-acting insulin composition for acute administration to a diabeticsubject in response to an actual, perceived, or anticipatedhyperglycemic condition in the subject arising at the time of, or withinabout four hours following, administration of the fast-acting insulin(such as a prandial hyperglycemic condition resulting or anticipated toresult from, consumption of a meal), whereby the fast-acting insulin isable to prevent, control or ameliorate the acute hyperglycemiccondition. Typically a fast-acting insulin composition exhibits peakinsulin levels at or about not more than four hours followingsubcutaneous administration to a subject. Fast-acting insulincompositions include recombinant insulins and isolated insulins (alsoreferred to as “regular” insulins) such as the insulin sold as Humulin®R, porcine insulins and bovine insulins, as well as insulin analogsdesigned to be rapid acting by virtue of amino acid changes. Exemplaryregular insulin preparations include, but are not limited to, humanregular insulins, such as those sold under the trademarks Humulin® R,Novolin® R and Velosulin®, Insulin Human, USP and Insulin HumanInjection, USP, as well as acid formulations of insulin, such as, forexample, Toronto Insulin, Old Insulin, and Clear Insulin, and regularpig insulins, such as Iletin II® (porcine insulin). Exemplary rapidacting insulin analogs include, for example, insulin lispro (e.g.Humalog® insulin), insulin aspart (e.g. NovoLog® insulin), and insulinglulisine (e.g. Apidra® insulin) the fast-acting insulin compositionsold as VIAject® and VIAtab® (see, e.g., U.S. Pat. No. 7,279,457). Whilethe term “fast-acting insulin” does not encompass “basal-actinginsulins,” the super fast-acting insulin compositions described hereinoptionally can include, in addition to a fast-acting insulin, one ormore basal-acting insulins.

As used herein, a human insulin refers to an insulin that is syntheticor recombinantly produced based upon the human polypeptide, includingallelic variants and analogs thereof.

As used herein, fast-acting human insulins or human fast-acting insulincompositions include any human insulin or composition of a human insulinthat is fast-acting, but excludes non-human insulins, such as regularpig insulin.

As used herein, the terms “basal-acting insulins,” or “basal insulins”refer to insulins administered to maintain a basal insulin level as partof an overall treatment regimen for treating a chronic condition suchdiabetes. Typically, a basal-acting insulin is formulated to maintain anapproximately steady state insulin level by the controlled release ofinsulin when administered periodically (e.g. once or twice daily).Basal-acting insulins include crystalline insulins (e.g. NPH and Lente®,protamine insulin, surfen insulin), basal insulin analogs (insulinglargine, HOE 901, NovoSol Basal) and other chemical formulations ofinsulin (e.g. gum arabic, lecithin or oil suspensions) that retard theabsorption rate of regular insulin. As used herein, the basal-actinginsulins can include insulins that are typically understood aslong-acting (typically reaching a relatively low peak concentration,while having a maximum duration of action over about 20-30 hours) orintermediate-acting (typically causing peak insulin concentrations atabout 4-12 hours after administration).

As used herein, “super fast-acting insulin composition” refers to aninsulin composition containing a fast-acting insulin and a hyaluronandegrading enzyme (such as a soluble hyaluronidase, including but notlimited to, rHuPH20 preparations), such that the insulin composition,over the first forty minutes following parenteral administration to asubject, provides a cumulative systemic insulin exposure in the subjectthat is greater than the cumulative systemic insulin exposure providedto the subject over the same period after administering the same dosageof the same fast-acting insulin, by the same route, in the absence ofthe hyaluronan degrading enzyme. The super fast-acting insulincomposition as described herein optionally can include a basal-actinginsulin.

As used herein, the terms “hyperglycemic condition” or “hyperglycemia”refer to an undesired elevation in blood glucose.

As used herein, the term “hypoglycemic condition” or “hypoglycemia”refers to an undesired drop in blood glucose.

As used herein, “systemic glucose clearance” or “systemic glucosemetabolism” refers to the removal of glucose from the blood and can beexpressed as either a rate (amount/time) or quantity (amount over aperiod of time). Systemic glucose clearance can be determined using anysuitable method known in the art. For example, systemic glucoseclearance can be measured using the Hyperinsulinemic-Euglycemic ClampProcedure under fasting conditions, such as that exemplified anddescribed herein, where the amount or rate of glucose infusedintravenously to maintain constant blood glucose levels, such as, forexample, 90-110 mg/dL, is equivalent to the systemic glucose clearance.The difference in the systemic glucose clearance achieved by differentinsulin compositions, such as the difference in the systemic glucoseclearance achieved by administration of a super fast-acting insulincomposition versus that achieved by a fast-acting insulin, can thereforebe determined using such procedures. The difference in systemic glucoseclearance among comparator insulins also can be determined by measuringthe relative glucose lowering activity of the comparator insulins at agiven point in time after a glucose challenge test. For example, aglucose challenge test (such as, for example, a 75-g oral glucosetolerance test or a standardized test meal formulation, well known tothose skilled in the art) can be used to compare different insulinpreparations. In such challenge tests, a quantity of glucose or othercarbohydrate is administered to a subject, immediately followed by anon-intravenous parenteral administration of the insulin composition.Blood glucose levels (i.e., concentration of glucose in the subject'sblood) is then measured at a predetermined time to determine the bloodlowering effect of the insulin. In these oral challenge comparisonsbetween various insulin preparations, the time elapsed after which theblood glucose levels are measured must be adequate to allow systemicglucose uptake. The studies described above to determine systemicglucose clearance can be performed using animal models and/or humansubjects.

As used herein, glycemic control or “controlling blood glucose levels”refers to the maintenance of blood glucose concentrations at a desiredlevel, typically between 70-130 mg/dL or 90-110 mg/dL.

As used herein, “cumulative systemic insulin exposure” or “cumulativesystemic exposure to insulin” refers to the amount of insulin that hasbeen absorbed into the blood following parenteral administration of theinsulin. Cumulative systemic exposure to insulin can be determined bycalculating the area under the curve for a specific period of time,where the curve is generated by plotting insulin concentration in theblood, serum or plasma as a function of time.

As used herein, a closed loop system is an integrated system forproviding continuous glycemic control. Closed loop systems contain amechanism for measuring blood glucose, a mechanism for delivering one ormore compositions, including an insulin composition, and a mechanism fordetermining the amount of insulin needed to be delivered to achieveglycemic control. Typically, therefore, closed loop systems contain aglucose sensor, an insulin delivery device, such as an insulin pump, anda controller that receives information from the glucose sensor andprovides commands to the insulin delivery device. The commands can begenerated by software in the controller. The software typically includesan algorithm to determine the amount of insulin required to be deliveredto achieve glycemic control, based upon the blood glucose levelsdetected by the glucose sensor or anticipated by the user.

As used herein, dosing regime refers to the amount of insulinadministered and the frequency of administration. The dosing regime is afunction of the disease or condition to be treated, and thus can vary.

As used herein, a hyaluronan degrading enzyme refers to an enzyme thatcatalyzes the cleavage of a hyaluronan polymer (also referred to ashyaluronic acid or HA) into smaller molecular weight fragments.Exemplary of hyaluronan degrading enzymes are hyaluronidases, andparticular chondroitinases and lyases that have the ability todepolymerize hyaluronan. Exemplary chondroitinases that are hyaluronandegrading enzymes include, but are not limited to, chondroitin ABC lyase(also known as chondroitinase ABC), chondroitin AC lyase (also known aschondroitin sulfate lyase or chondroitin sulfate eliminase) andchondroitin C lyase. Chondroitin ABC lyase comprises two enzymes,chondroitin-sulfate-ABC endolyase (EC 4.2.2.20) andchondroitin-sulfate-ABC exolyase (EC 4.2.2.21). An exemplarychondroitin-sulfate-ABC endolyases and chondroitin-sulfate-ABC exolyasesinclude, but are not limited to, those from Proteus vulgaris andFlavobacterium heparinum (the Proteus vulgaris chondroitin-sulfate-ABCendolyase is set forth in SEQ ID NO:98; Sato et al. (1994) Appl.Microbiol. Biotechnol. 41(1):39-46). Exemplary chondroitinase AC enzymesfrom the bacteria include, but are not limited to, those fromFlavobacterium heparinum Victivallis vadensis, set forth in SEQ IDNO:99, and Arthrobacter aurescens (Tkalec et al. (2000) Applied andEnvironmental Microbiology 66(1):29-35; Ernst et al. (1995) CriticalReviews in Biochemistry and Molecular Biology 30(5):387-444). Exemplarychondroitinase C enzymes from the bacteria include, but are not limitedto, those from Streptococcus and Flavobacterium (Hibi et al. (1989)FEMS-Microbiol-Lett. 48(2):121-4; Michelacci et al. (1976) J. Biol.Chem. 251:1154-8; Tsuda et al. (1999) Eur. J. Biochem. 262:127-133).

As used herein, hyaluronidase refers to a class of hyaluronan degradingenzymes. Hyaluronidases include bacterial hyaluronidases (EC 4.2.2.1 orEC 4.2.99.1), hyaluronidases from leeches, other parasites, andcrustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC3.2.1.35). Hyaluronidases include any of non-human origin including, butnot limited to, murine, canine, feline, leporine, avian, bovine, ovine,porcine, equine, piscine, ranine, bacterial, and any from leeches, otherparasites, and crustaceans. Exemplary non-human hyaluronidases include,hyaluronidases from cows (SEQ ID NOS:10, 11, 64 and BH55 (U.S. Pat. Nos.5,747,027 and 5,827,721), yellow jacket wasp (SEQ ID NOS:12 and 13),honey bee (SEQ ID NO:14), white-face hornet (SEQ ID NO:15), paper wasp(SEQ ID NO:16), mouse (SEQ ID NOS:17-19, 32), pig (SEQ ID NOS:20-21),rat (SEQ ID NOS:22-24, 31), rabbit (SEQ ID NO:25), sheep (SEQ ID NOS:26,27, 63 and 65), orangutan (SEQ ID NO:28), cynomolgus monkey (SEQ IDNO:29), guinea pig (SEQ ID NO:30), Arthrobacter sp. (strain FB24) (SEQID NO:67), Bdellovibrio bacteriovorus (SEQ ID NO:68), Propionibacteriumacnes (SEQ ID NO:69), Streptococcus agalactiae ((SEQ ID NO:70); 18RS21(SEQ ID NO:71); serotype Ia (SEQ ID NO:72); serotype III (SEQ ID NO:73),Staphylococcus aureus (strain COL) (SEQ ID NO:74); strain MRSA252 (SEQID NOS:75 and 76); strain MSSA476 (SEQ ID NO:77); strain NCTC 8325 (SEQID NO:78); strain bovine RF122 (SEQ ID NOS:79 and 80); strain USA300(SEQ ID NO:81), Streptococcus pneumoniae (SEQ ID NO:82); strain ATCCBAA-255/R6 (SEQ ID NO:83); serotype 2, strain D39/NCTC 7466 (SEQ IDNO:84), Streptococcus pyogenes (serotype (SEQ ID NO:85); serotype M2,strain MGAS10270 (SEQ ID NO:86); serotype M4, strain MGAS10750 (SEQ IDNO:87); serotype M6 (SEQ ID NO:88); serotype M12, strain MGAS2096 (SEQID NOS:89 and 90); serotype M12, strain MGAS9429 (SEQ ID NO:91);serotype M28 (SEQ ID NO:92); Streptococcus suis (SEQ ID NOS:93-95);Vibrio fischeri (strain ATCC 700601/ES114 (SEQ ID NO:96), and theStreptomyces hyaluronolyticus hyaluronidase enzyme, which is specificfor hyaluronic acid and does not cleave chondroitin or chondroitinsulfate (Ohya, T. and Kaneko, Y. (1970) Biochim. Biophys. Acta 198:607).Hyaluronidases also include those of human origin. Exemplary humanhyaluronidases include HYAL1 (SEQ ID NO:36), HYAL2 (SEQ ID NO:37), HYAL3(SEQ ID NO:38), HYAL4 (SEQ ID NO:39), and PH20 (SEQ ID NO:1). Alsoincluded amongst hyaluronidases are soluble hyaluronidases, including,ovine and bovine PH20, soluble human PH20 and soluble rHuPH20. Examplesof commercially available bovine or ovine soluble hyaluronidasesVitrase® (ovine hyaluronidase) and Amphadase® (bovine hyaluronidase).

Reference to hyaluronan degrading enzymes includes precursor hyaluronandegrading enzyme polypeptides and mature hyaluronan degrading enzymepolypeptides (such as those in which a signal sequence has beenremoved), truncated forms thereof that have activity, and includesallelic variants and species variants, variants encoded by splicevariants, and other variants, including polypeptides that have at least40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more sequence identity to the precursor polypeptides set forth inSEQ ID NOS: 1 and 10-48, 63-65, 67-100, or the mature form thereof. Forexample, reference to hyaluronan degrading enzyme also includes thehuman PH20 precursor polypeptide variants set forth in SEQ ID NOS:50-51.Hyaluronan degrading enzymes also include those that contain chemical orposttranslational modifications and those that do not contain chemicalor posttranslational modifications. Such modifications include, but arenot limited to, pegylation, albumination, glycosylation, farnesylation,carboxylation, hydroxylation, phosphorylation, and other polypeptidemodifications known in the art.

As used herein, a soluble hyaluronidase refers to a polypeptidecharacterized by its solubility under physiologic conditions. Solublehyaluronidases can be distinguished, for example, by its partitioninginto the aqueous phase of a Triton X-114 solution warmed to 37° C.(Bordier et al., (1981) J. Biol. Chem., 256:1604-7). Membrane-anchored,such as lipid anchored hyaluronidases, will partition into the detergentrich phase, but will partition into the detergent-poor or aqueous phasefollowing treatment with Phospholipase-C. Included among solublehyaluronidases are membrane anchored hyaluronidases in which one or moreregions associated with anchoring of the hyaluronidase to the membranehas been removed or modified, where the soluble form retainshyaluronidase activity. Soluble hyaluronidases include recombinantsoluble hyaluronidases and those contained in or purified from naturalsources, such as, for example, testes extracts from sheep or cows.Exemplary of such soluble hyaluronidases are soluble human PH20. Othersoluble hyaluronidases include ovine (SEQ ID NOS:27, 63, 65) and bovine(SEQ ID NOS:11, 64) PH20.

As used herein, soluble human PH20 or sHuPH20 include maturepolypeptides lacking all or a portion of the glycosylphospatidylinositol(GPI) attachment site at the C-terminus such that upon expression, thepolypeptides are soluble. Exemplary sHuPH20 polypeptides include maturepolypeptides having an amino acid sequence set forth in any one of SEQID NOS:4-9 and 47-48. The precursor polypeptides for such exemplarysHuPH20 polypeptides include a signal sequence. Exemplary of theprecursors are those set forth in SEQ ID NOS:3 and 40-46, each of whichcontains a 35 amino acid signal sequence at amino acid positions 1-35.Soluble HuPH20 polypeptides also include those degraded during or afterthe production and purification methods described herein.

As used herein, soluble recombinant human PH20 (rHuPH20) refers to asoluble form of human PH20 that is recombinantly expressed in ChineseHamster Ovary (CHO) cells. Soluble rHuPH20 is encoded by nucleic acidthat includes the signal sequence and is set forth in SEQ ID NO:49. Alsoincluded are DNA molecules that are allelic variants thereof and othersoluble variants. The nucleic acid encoding soluble rHuPH20 is expressedin CHO cells which secrete the mature polypeptide. As produced in theculture medium, there is heterogeneity at the C-terminus so that theproduct includes a mixture of species that can include any one or moreof SEQ ID NOS. 4-9 in various abundance. Corresponding allelic variantsand other variants also are included, including those corresponding tothe precursor human PH20 polypeptides set forth in SEQ ID NOS:50-51.Other variants can have 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ IDNOS:4-9 and 47-48 as long they retain a hyaluronidase activity and aresoluble.

As used herein, activity refers to a functional activity or activitiesof a polypeptide or portion thereof associated with a full-length(complete) protein. Functional activities include, but are not limitedto, biological activity, catalytic or enzymatic activity, antigenicity(ability to bind or compete with a polypeptide for binding to ananti-polypeptide antibody), immunogenicity, ability to form multimers,and the ability to specifically bind to a receptor or ligand for thepolypeptide.

As used herein, hyaluronidase activity refers to the ability toenzymatically catalyze the cleavage of hyaluronic acid. The UnitedStates Pharmacopeia (USP) XXII assay for hyaluronidase determineshyaluronidase activity indirectly by measuring the amount of highermolecular weight hyaluronic acid, or hyaluronan, (HA) substrateremaining after the enzyme is allowed to react with the HA for 30 min at37° C. (USP XXII-NF XVII (1990) 644-645 United States PharmacopeiaConvention, Inc, Rockville, Md.). A Reference Standard solution can beused in an assay to ascertain the relative activity, in units, of anyhyaluronidase. In vitro assays to determine the hyaluronidase activityof hyaluronidases, such as soluble rHuPH20, are known in the art anddescribed herein. Exemplary assays include the microturbidity assaydescribed below (see e.g. Example 3) that measures cleavage ofhyaluronic acid by hyaluronidase indirectly by detecting the insolubleprecipitate formed when the uncleaved hyaluronic acid binds with serumalbumin. Reference Standards can be used, for example, to generate astandard curve to determine the activity in Units of the hyaluronidasebeing tested.

As used herein, “functionally equivalent amount” or grammaticalvariations thereof, with reference to a hyaluronan degrading enzyme,refers to the amount of hyaluronan degrading enzyme that achieves thesame effect as an amount (such as a known number of Units ofhyaluronidase activity) of a reference enzyme, such as a hyaluronidase.For example, the activity of any hyaluronan degrading enzyme can becompared to the activity of rHuPH20 to determine the functionallyequivalent amount of a hyaluronan degrading enzyme that would achievethe same effect as a known amount of rHuPH20. For example, the abilityof a hyaluronan degrading enzyme to act as a spreading or diffusingagent can be assessed by injecting it into the lateral skin of mice withtrypan blue (see e.g. U.S. Pat. Publication No. 20050260186), and theamount of hyaluronan degrading enzyme required to achieve the sameamount of diffusion as, for example, 100 units of a HyaluronidaseReference Standard, can be determined. The amount of hyaluronandegrading enzyme required is, therefore, functionally equivalent to 100units. In another example, the ability of a hyaluronan degrading enzymeto increase the level and rate of absorption of a co-administeredinsulin can be assessed in human subjects, such as described below inExample 1, and the amount of hyaluronan degrading enzyme required toachieve the same increase in the level and rate of absorption of insulinas, for example, the administered quantity of rHuPH20, can be determined(such as by assessing the maximum insulin concentration in the blood(C_(max),) the time required to achieve maximum insulin concentration inthe blood (t_(max)) and the cumulative systemic insulin exposure over egiven period of time (AUC).

As used herein, the residues of naturally occurring α-amino acids arethe residues of those 20 α-amino acids found in nature which areincorporated into protein by the specific recognition of the chargedtRNA molecule with its cognate mRNA codon in humans.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, which are optionally labeled, such as with a detectable label,such as a fluorescent or radiolabel, single-stranded molecules arecontemplated. Such molecules are typically of a length such that theirtarget is statistically unique or of low copy number (typically lessthan 5, generally less than 3) for probing or priming a library.Generally a probe or primer contains at least 14, 16 or 30 contiguousnucleotides of sequence complementary to or identical to a gene ofinterest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleicacids long.

As used herein, a peptide refers to a polypeptide that is greater thanor equal to two amino acids in length, and less than or equal to 40amino acids in length.

As used herein, the amino acids which occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 1). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids, non-natural amino acids and amino acidanalogs (i.e., amino acids wherein the α-carbon has a side chain).

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are presumed to be inthe “L” isomeric form. Residues in the “D” isomeric form, which are sodesignated, can be substituted for any L-amino acid residue as long asthe desired functional property is retained by the polypeptide. NH₂refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), andadopted 37 C.F.R. □§§1.821-1.822, abbreviations for amino acid residuesare shown in Table 1:

TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is broadly defined to include the aminoacids listed in the Table of Correspondence (Table 1) and modified andunusual amino acids, such as those referred to in 37 C.F.R.§§1.821-1.822, and incorporated herein by reference. Furthermore, itshould be noted that a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues, to an amino-terminal group such as NH₂ orto a carboxyl-terminal group such as COOH.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides.

As used herein, “non-natural amino acid” refers to an organic compoundthat has a structure similar to a natural amino acid but has beenmodified structurally to mimic the structure and reactivity of a naturalamino acid. Non-naturally occurring amino acids thus include, forexample, amino acids or analogs of amino acids other than the 20naturally-occurring amino acids and include, but are not limited to, theD-isostereomers of amino acids. Exemplary non-natural amino acids aredescribed herein and are known to those of skill in the art.

As used herein, a DNA construct is a single- or double-stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and will be understood to beequivalent to the term base pairs. It will be recognized by thoseskilled in the art that the two strands of a double-strandedpolynucleotide can differ slightly in length and that the ends thereofcan be staggered; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will, ingeneral, not exceed 20 nucleotides in length.

As used herein, “similarity” between two proteins or nucleic acidsrefers to the relatedness between the sequence of amino acids of theproteins or the nucleotide sequences of the nucleic acids. Similaritycan be based on the degree of identity and/or homology of sequences ofresidues and the residues contained therein. Methods for assessing thedegree of similarity between proteins or nucleic acids are known tothose of skill in the art. For example, in one method of assessingsequence similarity, two amino acid or nucleotide sequences are alignedin a manner that yields a maximal level of identity between thesequences. “Identity” refers to the extent to which the amino acid ornucleotide sequences are invariant. Alignment of amino acid sequences,and to some extent nucleotide sequences, also can take into accountconservative differences and/or frequent substitutions in amino acids(or nucleotides). Conservative differences are those that preserve thephysico-chemical properties of the residues involved. Alignments can beglobal (alignment of the compared sequences over the entire length ofthe sequences and including all residues) or local (the alignment of aportion of the sequences that includes only the most similar region orregions).

“Identity” per se has an art-recognized meaning and can be calculatedusing published techniques. (See, e.g.: Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exists a number of methodsto measure identity between two polynucleotide or polypeptides, the term“identity” is well known to skilled artisans (Carrillo, H. & Lipton, D.,SIAM J Applied Math 48:1073 (1988)).

As used herein, homologous (with respect to nucleic acid and/or aminoacid sequences) means about greater than or equal to 25% sequencehomology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%,80%, 85%, 90% or 95% sequence homology; the precise percentage can bespecified if necessary. For purposes herein the terms “homology” and“identity” are often used interchangeably, unless otherwise indicated.In general, for determination of the percentage homology or identity,sequences are aligned so that the highest order match is obtained (see,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; Carrillo et al. (1988) SIAM J Applied Math 48:1073). By sequencehomology, the number of conserved amino acids is determined by standardalignment algorithms programs, and can be used with default gappenalties established by each supplier. Substantially homologous nucleicacid molecules would hybridize typically at moderate stringency or athigh stringency all along the length of the nucleic acid of interest.Also contemplated are nucleic acid molecules that contain degeneratecodons in place of codons in the hybridizing nucleic acid molecule.

Whether any two molecules have nucleotide sequences or amino acidsequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% “identical” or “homologous” can be determined using knowncomputer algorithms such as the “FASTA” program, using for example, thedefault parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.USA 85:2444 (other programs include the GCG program package (Devereux,J. et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN,FASTA (Altschul, S. F. et al., J Molec Biol 215:403 (1990)); Guide toHuge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994,and Carrillo et al. (1988) SIAM J Applied Math 48:1073). For example,the BLAST function of the National Center for Biotechnology Informationdatabase can be used to determine identity. Other commercially orpublicly available programs include, DNAStar “MegAlign” program(Madison, Wis.) and the University of Wisconsin Genetics Computer Group(UWG) “Gap” program (Madison Wis.). Percent homology or identity ofproteins and/or nucleic acid molecules can be determined, for example,by comparing sequence information using a GAP computer program (e.g.,Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith andWaterman (1981) Adv. Appl. Math. 2:482). Briefly, the GAP programdefines similarity as the number of aligned symbols (i.e., nucleotidesor amino acids), which are similar, divided by the total number ofsymbols in the shorter of the two sequences. Default parameters for theGAP program can include: (1) a unary comparison matrix (containing avalue of 1 for identities and 0 for non-identities) and the weightedcomparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, asdescribed by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE ANDSTRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979);(2) a penalty of 3.0 for each gap and an additional 0.10 penalty foreach symbol in each gap; and (3) no penalty for end gaps.

Therefore, as used herein, the term “identity” or “homology” representsa comparison between a test and a reference polypeptide orpolynucleotide. As used herein, the term at least “90% identical to”refers to percent identities from 90 to 99.99 relative to the referencenucleic acid or amino acid sequence of the polypeptide. Identity at alevel of 90% or more is indicative of the fact that, assuming forexemplification purposes a test and reference polypeptide length of 100amino acids are compared. No more than 10% (i.e., 10 out of 100) of theamino acids in the test polypeptide differs from that of the referencepolypeptide. Similar comparisons can be made between test and referencepolynucleotides. Such differences can be represented as point mutationsrandomly distributed over the entire length of a polypeptide or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g. 10/100 amino acid difference (approximately 90%identity). Differences are defined as nucleic acid or amino acidsubstitutions, insertions or deletions. At the level of homologies oridentities above about 85-90%, the result should be independent of theprogram and gap parameters set; such high levels of identity can beassessed readily, often by manual alignment without relying on software.

As used herein, an aligned sequence refers to the use of homology(similarity and/or identity) to align corresponding positions in asequence of nucleotides or amino acids. Typically, two or more sequencesthat are related by 50% or more identity are aligned. An aligned set ofsequences refers to 2 or more sequences that are aligned atcorresponding positions and can include aligning sequences derived fromRNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

As used herein, “primer” refers to a nucleic acid molecule that can actas a point of initiation of template-directed DNA synthesis underappropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and a polymerization agent, such as DNApolymerase, RNA polymerase or reverse transcriptase) in an appropriatebuffer and at a suitable temperature. It will be appreciated that acertain nucleic acid molecules can serve as a “probe” and as a “primer.”A primer, however, has a 3′ hydroxyl group for extension. A primer canbe used in a variety of methods, including, for example, polymerasechain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR,multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′ and 5′RACE, in situ PCR, ligation-mediated PCR and other amplificationprotocols.

As used herein, “primer pair” refers to a set of primers that includes a5′ (upstream) primer that hybridizes with the 5′ end of a sequence to beamplified (e.g. by PCR) and a 3′ (downstream) primer that hybridizeswith the complement of the 3′ end of the sequence to be amplified.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. anoligonucleotide) to a target nucleic acid molecule. Those of skill inthe art are familiar with in vitro and in vivo parameters that affectspecific hybridization, such as length and composition of the particularmolecule. Parameters particularly relevant to in vitro hybridizationfurther include annealing and washing temperature, buffer compositionand salt concentration. Exemplary washing conditions for removingnon-specifically bound nucleic acid molecules at high stringency are0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1%SDS, 50° C. Equivalent stringency conditions are known in the art. Theskilled person can readily adjust these parameters to achieve specifichybridization of a nucleic acid molecule to a target nucleic acidmolecule appropriate for a particular application. Complementary, whenreferring to two nucleotide sequences, means that the two sequences ofnucleotides are capable of hybridizing, typically with less than 25%,15% or 5% mismatches between opposed nucleotides. If necessary, thepercentage of complementarity will be specified. Typically the twomolecules are selected such that they will hybridize under conditions ofhigh stringency.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, it also is understood that the terms “substantiallyidentical” or “similar” varies with the context as understood by thoseskilled in the relevant art.

As used herein, an allelic variant or allelic variation references anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and can result in phenotypic polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or canencode polypeptides having altered amino acid sequence. The term“allelic variant” also is used herein to denote a protein encoded by anallelic variant of a gene. Typically the reference form of the geneencodes a wildtype form and/or predominant form of a polypeptide from apopulation or single reference member of a species. Typically, allelicvariants, which include variants between and among species typicallyhave at least 80%, 90% or greater amino acid identity with a wildtypeand/or predominant form from the same species; the degree of identitydepends upon the gene and whether comparison is interspecies orintraspecies. Generally, intraspecies allelic variants have at leastabout 80%, 85%, 90% or 95% identity or greater with a wildtype and/orpredominant form, including 96%, 97%, 98%, 99% or greater identity witha wildtype and/or predominant form of a polypeptide. Reference to anallelic variant herein generally refers to variations in proteins amongmembers of the same species.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide or several nucleotides, and caninclude modifications such as substitutions, deletions and insertions ofnucleotides. An allele of a gene also can be a form of a gene containinga mutation.

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, modification is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements of amino acids and nucleotides, respectively. Methods ofmodifying a polypeptide are routine to those of skill in the art, suchas by using recombinant DNA methodologies.

As used herein, the term promoter means a portion of a gene containingDNA sequences that provide for the binding of RNA polymerase andinitiation of transcription. Promoter sequences are commonly, but notalways, found in the 5′ non-coding region of genes.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

The term substantially free of cellular material includes preparationsof proteins in which the protein is separated from cellular componentsof the cells from which it is isolated or recombinantly-produced. In oneembodiment, the term substantially free of cellular material includespreparations of enzyme proteins having less that about 30% (by dryweight) of non-enzyme proteins (also referred to herein as acontaminating protein), generally less than about 20% of non-enzymeproteins or 10% of non-enzyme proteins or less that about 5% ofnon-enzyme proteins. When the enzyme protein is recombinantly produced,it also is substantially free of culture medium, i.e., culture mediumrepresents less than about or at 20%, 10% or 5% of the volume of theenzyme protein preparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of enzyme proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of enzyme proteins having less than about 30% (by dryweight) 20%, 10%, 5% or less of chemical precursors or non-enzymechemicals or components.

As used herein, synthetic, with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce a heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vehiclesare well known to those of skill in the art.

As used herein, an expression vector includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, vector also includes “virus vectors” or “viral vectors.”Viral vectors are engineered viruses that are operatively linked toexogenous genes to transfer (as vehicles or shuttles) the exogenousgenes into cells.

As used herein, operably or operatively linked when referring to DNAsegments means that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiatesdownstream of the promoter and upstream of any transcribed sequences.The promoter is usually the domain to which the transcriptionalmachinery binds to initiate transcription and proceeds through thecoding segment to the terminator.

As used herein, the term assessing is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of a protease, or a domain thereof, present inthe sample, and also of obtaining an index, ratio, percentage, visual orother value indicative of the level of the activity. Assessment can bedirect or indirect and the chemical species actually detected need notof course be the proteolysis product itself but can for example be aderivative thereof or some further substance. For example, detection ofa cleavage product of a complement protein, such as by SDS-PAGE andprotein staining with Coomasie blue.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein a biological activity of aprotease is its catalytic activity in which a polypeptide is hydrolyzed.

As used herein equivalent, when referring to two sequences of nucleicacids, means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with only aminoacid substitutions that do not substantially alter the activity orfunction of the protein or peptide. When equivalent refers to aproperty, the property does not need to be present to the same extent(e.g., two peptides can exhibit different rates of the same type ofenzymatic activity), but the activities are usually substantially thesame.

As used herein, “modulate” and “modulation” or “alter” refer to a changeof an activity of a molecule, such as a protein. Exemplary activitiesinclude, but are not limited to, biological activities, such as signaltransduction. Modulation can include an increase in the activity (i.e.,up-regulation or agonist activity), a decrease in activity (i.e.,down-regulation or inhibition) or any other alteration in an activity(such as a change in periodicity, frequency, duration, kinetics or otherparameter). Modulation can be context dependent and typically modulationis compared to a designated state, for example, the wildtype protein,the protein in a constitutive state, or the protein as expressed in adesignated cell type or condition.

As used herein, a composition refers to any mixture. It can be asolution, suspension, liquid, powder, paste, aqueous, non-aqueous or anycombination thereof.

As used herein, a combination refers to any association between or amongtwo or more items. The combination can be two or more separate items,such as two compositions or two collections, can be a mixture thereof,such as a single mixture of the two or more items, or any variationthereof. The elements of a combination are generally functionallyassociated or related.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms. Diseases and disorders ofinterest herein are those involving components of the ECM.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment. Hence treatment encompassesprophylaxis, therapy and/or cure. Prophylaxis refers to prevention of apotential disease and/or a prevention of worsening of symptoms orprogression of a disease. Treatment also encompasses any pharmaceuticaluse of a super fast-acting insulin composition provided herein.

As used herein, a pharmaceutically effective agent, includes anytherapeutic agent or bioactive agents, including, but not limited to,for example, anesthetics, vasoconstrictors, dispersing agents,conventional therapeutic drugs, including small molecule drugs andtherapeutic proteins.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease or other indication, are ameliorated orotherwise beneficially altered.

As used herein, a therapeutic effect means an effect resulting fromtreatment of a subject that alters, typically improves or amelioratesthe symptoms of a disease or condition or that cures a disease orcondition. A therapeutically effective amount refers to the amount of acomposition, molecule or compound which results in a therapeutic effectfollowing administration to a subject.

As used herein, the term “subject” refers to an animal, including amammal, such as a human being.

As used herein, a patient refers to a human subject exhibiting symptomsof a disease or disorder.

As used herein, amelioration of the symptoms of a particular disease ordisorder by a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms that canbe attributed to or associated with administration of the composition ortherapeutic.

As used herein, prevention or prophylaxis refers to methods in which therisk of developing disease or condition is reduced.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect. Hence, it is thequantity necessary for preventing, curing, ameliorating, arresting orpartially arresting a symptom of a disease or disorder.

As used herein, a therapeutically effective insulin dosage is the amountof insulin required or sufficient to achieve glycemic control. Thisamount can be determined empirically, such as by glucose or mealchallenge. The compositions provided herein contain a therapeuticallyeffective amount or concentration of insulin so that therapeuticallyeffective dosages are administered.

As used herein, unit dose form refers to physically discrete unitssuitable for human and animal subjects and packaged individually as isknown in the art.

As used herein, a single dosage formulation refers to a formulation fordirect administration.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass a fast-acting insulin composition and hyaluronan degradingenzyme composition contained in the same or separate articles ofpackaging.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a “kit” refers to a combination of compositions providedherein and another item for a purpose including, but not limited to,reconstitution, activation, instruments/devices for delivery,administration, diagnosis, and assessment of a biological activity orproperty. Kits optionally include instructions for use.

As used herein, a cellular extract or lysate refers to a preparation orfraction which is made from a lysed or disrupted cell.

As used herein, animal includes any animal, such as, but are not limitedto primates including humans, gorillas and monkeys; rodents, such asmice and rats; fowl, such as chickens; ruminants, such as goats, cows,deer, sheep; ovine, such as pigs and other animals. Non-human animalsexclude humans as the contemplated animal. The enzymes provided hereinare from any source, animal, plant, prokaryotic and fungal. Most enzymesare of animal origin, including mammalian origin.

As used herein, a control refers to a sample that is substantiallyidentical to the test sample, except that it is not treated with a testparameter, or, if it is a plasma sample, it can be from a normalvolunteer not affected with the condition of interest. A control alsocan be an internal control.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a compound, comprising “an extracellular domain”includes compounds with one or a plurality of extracellular domains.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochemistry11:1726).

B. SUPER FAST-ACTING INSULIN COMPOSITIONS

Provided herein are super fast-acting insulin combinations andcompositions. The super fast-acting insulin compositions are obtained bycombining, before, or at the time of administration, a fast-actinginsulin and a hyaluronan degrading enzyme. Also provided are methods anduses of the super fast-acting insulin composition to treat the samediseases and conditions for which fast-acting insulins have heretoforebeen indicated, for example, diabetes mellitus for the control ofhyperglycemia and other diseases and conditions. Fast-acting insulins(for example Humalog® insulin lispro and Humulin® R insulin) do notadequately mimic the endogenous insulin spike of the first phaseprandial insulin release. It is now discovered that by combining afast-acting insulin with a hyaluronan degrading enzyme, the methods,compositions and combinations described herein provide a superfast-acting insulin composition that more closely mimics the endogenous(i.e., natural) post-prandial insulin release of a nondiabetic subject.

1. Overview of Insulin, Diabetes and Existing Fast-Acting InsulinTherapies

Insulin is a naturally-occurring polypeptide hormone secreted by thepancreas. Insulin is required by the cells of the body to effectivelytake up and use glucose from the blood. Glucose is the predominantenergy substrate to carry out cellular functions. In addition to beingthe primary modulator of carbohydrate homeostasis, insulin has effectson fat metabolism. It can change the ability of the liver and adiposetissue, among others, to release fat stores. Insulin has variouspharmacodynamic effects throughout the body, including but not limitedto increase in lipid synthesis, reduction in lipid breakdown, increasein protein synthesis, regulation of key enzymes and processes in glucosemetabolism (including glucose uptake stimulation, glucose oxidationstimulation, increased glycogen synthesis and reduced glycogenbreakdown).

Although insulin is secreted basally, usually in the range of 0.5 to 1.0unit per hour, its levels are increased after a meal. After a meal, thepancreas secretes a bolus of insulin in response to a rise in glucose.Insulin stimulates the uptake of glucose into cells, and signals theliver to reduce glucose production; this results in a return of bloodglucose to normal levels. In normal adults, there are two phases ofinsulin release in response to a meal. The early phase is a spike ofinsulin release that occurs within 2-15 minutes of eating. The latephase release extends about 2 hours. The early phase is responsible forshutting down hepatic glucose production, thereby reducing blood glucoselevels and sensitizing or signaling peripheral tissues to increaseglucose uptake. In muscle, large amounts of glucose are stored asglycogen. Some of the glycogen is broken down into lactate, whichcirculates to the liver and can be converted back into glucose andstored as glycogen. Between meals the liver breaks down these glycogenstores to provide glucose to the brain and other tissues.

Diabetes results in chronic hyperglycemia due to the inability orreduced ability of the pancreas to produce adequate amounts of insulinor due to the inability or reduced ability of cells to synthesize and/orrelease the insulin required. In diabetics, the effectiveness of theabove described first-phase response is decreased or absent, leading toelevated postprandial glucose levels. For example, blood glucose areaunder the curve (AUC) during the first four postprandial hours (i.e.first four hours after eating), is 2.5 to 3.0 times greater in diabeticsthan in non-diabetics. Postprandial glucose excursions contribute tooverall hyperglycemia and elevated HbAlc levels, and these excursionsare the primary contributors to HbAlc elevations seen in early stages ofType 2 diabetes.

Many diabetic patients require treatment with insulin when the pancreasproduces inadequate amounts of insulin, or cannot use the insulin itproduces, to maintain adequate glycemic control. Insulin has beenadministered as a therapeutic to treat patients having diabetes,including, for example, type 1 diabetes, type 2 diabetes and gestationaldiabetes, in order to mimic the endogenous insulin response that occursin normal individuals. Insulin also has been administered to criticallyill patients with hyperglycemia to control blood glucose levels.Different sources of insulins are used depending on the patient need.Commercial insulin preparations can be classified depending on theirduration of activity (see e.g., DeFelippis et al. (2002) InsulinChemistry and Pharmacokinetics. In Ellenberg and Rifkin's DiabetesMellitus (pp. 481-500) McGraw-Hill Professional). For example, insulinis provided in fast-acting formulations, as well as intermediate- orlong-acting formulations, the latter two classifications being referredto herein as basal-acting insulins. The fast-acting forms have a rapidonset, typically exhibiting peak insulin levels in 2-3 hours or less,and no more than four hours. Hence, fast-acting forms of insulin areused in prandial glucose regulation. Other forms of insulin includeintermediate-acting, which reach peak insulin concentration atapproximately 4-12 hours following subcutaneous administration, andlong-acting insulins that reach a relatively modest peak and have amaximum duration of action of 20-30 hours. The intermediate- andlong-acting forms are often composed of amorphous and/or crystallineinsulin preparations, and are used predominantly in basal therapies.

The goal of prandial administration of fast-acting insulin compositionsis to attain a stable blood glucose level over time by parenteraladministration of the fast-acting insulin before, during or soon aftermealtime. In this way, blood levels of insulin are temporarily elevatedto (a) shut down hepatic glucose production and (b) increase glucoseuptake; thus maintaining glycemic control during the elevation in bloodglucose associated with meal digestion.

Recombinant human insulin (e.g., Humulin® R insulin) is used for selfadministration by injection prior to meal time. Unfortunately,recombinant human insulin must be dosed by injection approximately onehalf hour or more prior to meal time in order to insure that a rise inblood glucose does not occur unopposed by exogenous insulin levels. Oneof the reasons for the slow absorption of recombinant human insulin isbecause insulin forms hexameric complexes in the presence of zinc ionsboth in vivo and in vitro. Such hexameric zinc-containing complexes aremore stable than monomeric insulin lacking zinc. Upon subcutaneousinjection, these insulin hexamers must dissociate into smaller dimers ormonomers before they can be absorbed through capillary beds and passinto the systemic circulation. The dissociation of hexamers to dimersand monomers is concentration-dependent, occurring only at lowerconcentrations as the insulin diffuses from the injection site. Thus, alocal insulin depot exists at the injection site following subcutaneousadministration of insulin, providing an initial high concentration ofhexameric insulin at the site of injection that can not be absorbeduntil the insulin concentration decreases (Soeborg et al., (2009) Eur.J. Pharm. Sci. 36:78-90). As the insulin slowly diffuses from theinjection site, the insulin concentration lowers as the distance fromthe injection site increases, resulting in dissociation of the hexamersand absorption of the insulin monomers and dimers. Thus, althoughdispersal of hexameric insulin complexes occurs naturally in the body,it can take some time to occur, delaying the systemic availability ofinsulin. Further, because of this concentration-dependent absorption,higher insulin concentrations and higher doses and are absorbed moreslowly (Soeborg et al., (2009) Eur. J. Pharm. Sci. 36:78-90).

Since insulin in monomeric form is absorbed more rapidly, while insulinsin the hexameric state are more stable, fast-acting analog forms ofinsulin have been developed that exhibit a faster dissociation fromhexameric to monomeric upon subcutaneous administration. Such insulinsare modified, such as by amino acid change, to increase the dissociationrate, thereby imparting more rapid pharmacodynamic activity uponinjection. As described in Section C, fast-acting analog forms ofinsulin include but are not limited to, insulin glulisine, insulinaspart, and insulin lispro.

Fast-acting forms of insulins, including fast-acting analogs, have adelay in absorption and action, and therefore do not approximateendogenous insulin that has a early phase that occurs about 10 minutesafter eating. Thus, such formulations do not act quickly enough to shutoff hepatic glucose production that occurs shortly after this firstphase of insulin release. For this reason, even the fast-acting insulinanalog preparations must be given in advance of meals in order toachieve any chance of desired glycemic control. Although it is easier toestimate time of eating within 15 minutes than within 30-60 minutesrequired for regular insulin, there is a risk that a patient may eat tooearly or too late to provide the best blood glucose control.

Further, one of the main side effects of treatment with any insulintherapy, including fast-acting insulin therapies, is hypoglycemia.Hypoglycemia is defined as low blood glucose and is associated with avariety of morbidities that may range from, hunger to more bothersomesymptoms such as tremor, sweating, confusion or all the way to seizure,coma and death. Hypoglycemia can occur from failure to eat enough,skipping meals, exercising more then usual or taking too much insulin orusing an prandial insulin preparation that has an inappropriately longduration of exposure and action. For example, since many fast-actinginsulin therapies must be given before a meal, there is a risk that apatient may forego or skip the meal, leading to hypoglycemia.Additionally, upon administration of a fast-acting insulin, seruminsulin levels and insulin action (measured, for example, as glucoseinfusion rate (GIR)) typically remain elevated after the prandialglucose load has abated, threatening hypoglycemia. Attempts to bettercontrol peak glucose loads by increasing insulin dose further increasesthis danger. Also, because postprandial hypoglycemia is a common resultof insulin therapy, it often causes or necessitates that patients eatsnacks between meals. This contributes to the weight gain and obesityoften associated with insulin therapies.

2. Pharmacodynamics and Pharmacokinetics of a Super Fast-Acting InsulinComposition

It is discovered herein that the combination of a fast-acting insulinand a hyaluronan degrading enzyme results in an increased absorption ofthe fast-acting insulin, resulting in a more rapid rise in serum insulinconcentration (i.e. more rapid rate of absorption) and pharmacologicalaction. Hence, the combination of a fast-acting insulin and a hyaluronandegrading enzyme results in a super fast-acting insulin compositioncapable of effecting a rapid rise in blood glucose following parenteral(i.e., non intravenous) bolus administration (such as for exampleparenteral administration via subcutaneous (SC), intramuscular (IM),intraperitoneal (IP), or intradermal (ID) routes of administration.

While not being bound by any theory, the combination of a fast-actinginsulin and a hyaluronan degrading enzyme can result in increasedabsorption of the fast-acting insulin, compared to when the insulin isadministered alone, due to a change in the mechanism of dispersionfollowing subcutaneous administration. Typically, the presence of highmolecular weight hyaluronan provides a barrier to the flow of bulk fluidfollowing subcutaneous injection of insulin alone. Thus, as discussedabove, the insulin is dispersed from the site of injection bydiffusion-mediated mechanisms. As the insulin disperses from the site ofinjection, the concentration decreases, facilitating dissociation ofinsulin hexamers to monomers and dimers, which are small enough to beabsorbed through the capillary beds. Thus, to be absorbed followingsubcutaneous injection, the insulin must first slowly disperse from thesite of injection to create the sufficiently low insulin concentrationsto facilitate dissociation and, therefore, absorption. However, when theinsulin is co-administered with a hyaluronan-degrading enzyme, such as,for example, a soluble hyaluronidase, the hyaluronan is degraded by thehyaluronan-degrading enzyme, enabling the flow of bulk fluid, which israpidly dispersed proportional to the pressure gradient (or hydraulicconductivity). At physiologic pressure, for example, a solublehyaluronidase such as rHuPH20 generates an approximate 20-fold increasein hydraulic conductance. Thus, when co-administered with ahyaluronan-degrading enzyme, the insulin is rapidly dispersed in aconvection-mediated manner following degradation of the hyaluronanbarrier. This rapid absorption of insulin when co-administeredsubcutaneously with hyaluronan degrading enzyme can result in improvedpharmacokinetic and pharmacodynamic properties of the insulin comparedto when the insulin is administered alone.

For example, as provided herein, the super fast-acting insulincomposition is absorbed faster as demonstrated by a reduction oft_(max), and increased C_(max) and cumulative systemic insulin exposurethat is especially pronounced over the first 40 minutes. This improvedpharmacokinetic profile is reflected in a shortened onset and durationof insulin effect. This can be exemplified by pharmacodynamic measures,such as by glucose infusion rates in euglycemic clamp experiments suchas is described in Example 1. Thus, a super fast-acting insulincomposition is more rapidly absorbed than the corresponding fast-actinginsulin. Interestingly, as set forth in FIGS. 1 and 2, the superfast-acting insulin compositions containing a hyaluronan degradingenzyme exhibit an accelerated absorption of both fast-acting regularinsulins and fast-acting insulin analogs resulting in similarpharmacodynamic (PD) and pharmacokinetic (PK) profiles, even thoughfast-acting insulin analog is substantially faster than the fast-actingregular insulin without the hyaluronan degrading enzyme. Thus, superfast-acting insulin compositions exhibit similar pharmacodynamic (PD)and pharmacokinetic (PK) profiles, regardless of whether a fast-actinginsulin analog or fast-acting regular insulin is included in thecomposition. This similarity is particularly striking in the first 40 to60 minutes following administration (see e.g., FIGS. 1 and 2). Hence, anadditional advantage of the super fast-acting insulin composition is theability to achieve comparable pharmacokinetic and pharmacodynamicprofiles in the first 40 to 60 minutes following administration, withoutregard to whether the fast-acting insulin is a fast-acting regularinsulin (e.g., Humulin® R insulin) or a fast-acting analog (such asHumalog® insulin lispro, Novalog® insulin aspart or Apidra® insulinglulisine). In some instances, such as where the fast-acting insulin inthe super fast-acting insulin composition is a rapid acting insulinanalog, rather than a regular insulin, the absorption of the fast actinginsulin when administered with the hyaluronan degrading enzyme (i.e. asa super fast acting insulin composition) is faster than the fastest ofthe fast acting insulins alone. This can manifest itself as, forexample, decreased t_(max) and increased cumulative systemic insulinexposure, particularly over the first 40 minutes.

The pharmacokinetics of the super fast-acting insulin compositiondiffers from the corresponding fast-acting insulin in several importantrespects. First, the profile of insulin blood concentration as afunction of time is shifted to one of higher concentrations at earliertimes (see for example, FIG. 1). This rate of appearance of insulin intothe systemic circulation is described as the absorption rate, asdistinguished from the rate of removal from the systemic circulation,which is described as the clearance rate. Super fast-acting insulincompositions have a greater absorption rate, resulting in greater earlyexposure, than the corresponding fast-acting insulin. Moreover, becausethe hyaluronan degrading enzyme is transiently and locally acting at thesite of administration, the clearance rate of the super fast-actinginsulin composition and its potency once in the systemic circulation arenot materially different from the corresponding fast-acting insulin. Byincreasing the absorption rate while maintaining the same clearancerate, the maximum blood concentration of insulin (C_(max)) also isincreased for a super fast-acting insulin composition relative to thecorresponding fast-acting insulin. Thus, the same total quantity ofsystemically available insulin is distributed differently as a functionof time for a super fast-acting insulin composition relative to thecorresponding fast-acting insulin, such that, following parenteraladministration of a super fast-acting insulin composition, a greaterfraction of the cumulative systemic insulin exposure occurs over earliertime points and a smaller fraction of the cumulative systemic insulinexposure occurs over later time points, as compared to an insulin thatis merely fast-acting. This shift in the absorption rate enables thesuper fast-acting insulin composition to more closely mimic the body'sendogenous insulin response to the spike in blood glucose levels thatoccurs after consumption of a meal.

A second and independent pharmacokinetic parameter, the fraction of theadministered dose that reaches the systemic circulation, also can differfrom the super fast-acting insulin composition relative to itscorresponding fast-acting insulin. For certain fast-acting insulins, thevast majority of the administered dose is systemically bioavailable, andhence there may only be an incremental increase for the correspondingsuper fast-acting composition. However for other fast-acting insulins,such as regular insulin (for example Humulin® R insulin), the increasein bioavailability can be significant. The relative bioavailability of asuper fast-acting insulin composition as described herein to itscorresponding fast-acting insulin is described by the ratio of the totalsystemic exposure (AUC_(0-infinity)) of the two compositions followingidentical non-IV parenteral administrations.

A further important aspect of the super fast-acting insulin compositionsconcerns the ability to achieve improvement in pharmacodynamicparameters that measure the physiological response to the systemicallyavailable insulin. Because the super fast-acting insulin compositionsdescribed herein have the same pharmacological potency upon reaching thesystemic circulation as the corresponding fast-acting insulin, theimproved pharmacokinetic profiles offered by the super fast-actinginsulin compositions (as discussed above) result in beneficial changesin pharmacodynamic parameters that measure the physiological response tothe systemically available insulin. For example, the glucose infusionrate (GIR) measured when insulin is administered to subjects in aeuglycemic clamp procedure represents a pharmacodynamic parameter as itmeasures the rate of intravenous glucose administration as a function oftime required to maintain a steady target blood glucose concentration.By virtue of the pharmacokinetic advantage of greater absorption rateachieved by a super fast-acting insulin composition compared to thecorresponding fast-acting insulin, the super fast-acting insulincomposition is able to shift the GIR profile (a measure of thephysiological response to the insulin) toward greater infusion rates(i.e., greater physiological response) at earlier times. For those superfast-acting insulin compositions where there also is a meaningfulincrease in relative systemic bioavailability, a further increase in GIRresponse can be observed, although the total GIR is a function of boththe distribution of insulin levels as a function of time and of thesystemic dose administered.

The pharmacokinetic and pharmacodynamic advantages afforded by the superfast-acting insulin compositions described herein lead to a number ofimportant uses. First, by shifting the PK and PD responses to earliertimes, a more natural insulin response for the super fast-actingcomposition can be produced to control postprandial glucose levels thanis possible with the corresponding fast-acting insulin compositionalone. The body's natural insulin response includes both (a) an initialburst of insulin within the first 10-15 minutes signaling shutdown ofthe hepatic glucose release and providing minimum glucose bloodconcentrations between meals; and (b) a total insulin exposure overabout 2 hours, which is matched to the carbohydrate composition of themeal by adjusting insulin release into the systemic circulation as afunction of glucose levels through a complex interplay of hormonalresponses, including both beta cell responses to systemic metabolite(predominately glucose) levels and incretin hormones which potentiateinsulin secretion when the intestinal tract senses the presence ofnutrient materials. By having a greater fraction of the systemicallyavailable insulin exposure occur over the first 10-15 minutes, the superfast-acting insulin composition is better able to signal the shutdown ofthe hepatic glucose release (like the body's endogenous prandial insulinresponse) as compared to the corresponding fast-acting insulincomposition. Moreover, the super fast-acting insulin compositionsdescribed herein also are better able to mimic the natural control ofpost-prandial glucose, by having a greater fraction of the systemicallyavailable insulin exposure over the first 2 hours and a correspondingreduction in the insulin exposure after 2 hours. Elevated insulin levelsoccurring more than 2 hours after administration can result in anincreased glucose metabolism when postprandial glucose absorption iscomplete, a situation that leads to low blood glucose levels orhypoglycemia. Additionally, because the super fast-acting insulincompositions have an onset of action similar to the natural insulinresponse, these compositions can be administered at mealtime, while manyfast-acting insulin compositions (for example, Humulin® R insulin) areadministered 30-60 minutes prior to a meal, which introduces a risk ofhypoglycemia if the subject delays or skips the intended meal. Thus,through the combination of increased insulin exposure over the first 15minutes, and decreased insulin exposure after 2 hours, super fast-actinginsulin compositions are better able to control postprandial glucoselevels than the corresponding fast-acting insulin compositions.

Fast-acting insulins typically are administered over a wide range ofdoses as determined by the physician or other qualified healthcareprovider depending on many factors including the actual glucose levels,the subject, the type of diabetes and the composition of the meal.Typically, such fast-acting insulin doses can be in the range of between0.05 Units/kg to 2 Units/kg. Due to their pharmacokinetics andpharmacodynamics, super fast-acting insulin compositions can beadministered at lower doses compared to the fast-acting insulinadministered in the absence of a hyaluronan degrading enzyme. The degreeto which the amount of a fast-acting insulin can be lowered byadministering it as a super fast-acting insulin composition varies,depending on, for example, the type of diabetes the patient has.Typically, the reduction in the amount of fast-acting insulinadministered to Type 2 diabetic patients when administered as a superfast-acting insulin composition is greater than the reduction in theamount of fast-acting insulin administered to Type 1 diabetic patientswhen administered as a super fast-acting insulin composition. Forexample, in instances where a Type 1 diabetic patient and Type 2diabetic patient are each administered 0.20 U/kg of fast-acting insulinto control postprandial glucose levels, the Type 1 diabetic patient canbe administered 0.15 U/kg of fast-acting insulin in a super fast-actinginsulin composition to achieve the same or better glycemic control, andthe Type 2 diabetic patient can be administered 0.10 U/kg fast-actinginsulin in a super fast-acting insulin composition to achieve the sameor better glycemic control. Thus, in some examples, it is contemplatedherein that the amount of a fast-acting insulin that is administered toa Type 2 diabetic patient to achieve glycemic control can be reduced by,for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80% or more when administered with a hyaluronan degradingenzyme as a super fast-acting insulin composition compared to the amountrequired for glycemic control when administered without a hyaluronandegrading enzyme, and that the amount of a fast-acting insulin that isadministered to a Type 1 diabetic patient to achieve glycemic controlcan be reduced by, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70% or more when administered with a hyaluronandegrading enzyme as a super fast-acting insulin composition compared tothe amount required for glycemic control when administered without ahyaluronan degrading enzyme.

While not being bound by any theory, the greater reduction in thefast-acting insulin dose for Type 2 diabetic patients compared to Type 1diabetic patients when the insulin is administered with a hyaluronandegrading enzyme as a super fast-acting insulin composition is areflection of the different postprandial glycemic profiles of Type 1 andType 2 patients, and the ability of the super fast-acting insulin tomore closely mimic the natural first phase insulin release in healthysubjects. Type 2 diabetes develops as a result of impaired β cellfunction, insulin resistance, and/or impaired insulin secretion. Thesepatients lack the early phase insulin release that occurs within minutesof a glucose challenge, such as a meal, but still slowly release insulinover time. In contrast, Type 1 diabetic patients do not produce anyinsulin and so lack both the first and second phase insulin release, thelatter of which is sustained in healthy subjects until glycemic controlis achieved. Thus, because Type 2 diabetics generally only requireinsulin therapy primarily to address post-prandial hyperglycemia, aproblem to be overcome in prandial insulin therapy in such diabetics isthe occurrence of hypoglycemia. Hypoglycemia can result if the subject'sown delayed and/or basal insulin secretion is coupled with the glucoselowering effect of any excess exogenous insulin remaining after theprandial spike has been alleviated. Over time, repeated occurrences ofsuch post-prandial hypoglycemic episodes can contribute to weight gainand obesity. The pharmacokinetics and pharmacodynamics of thefast-acting insulins are such that the dose that is needed to achieve anappropriate concentration of insulin in the blood quickly enough tolower glucose levels immediately following digestion of a meal (i.e. adose that covers the natural early phase insulin release) is one thatresults in excess insulin circulating in the blood following digestionand lowering of the postprandial glucose levels. Therefore, Type 2diabetic patients receive insulin doses that cover more than just theearly phase insulin release. The super fast-acting insulin compositionsprovided herein more closely mimic the endogenous insulin response.Thus, Type 2 diabetics can be administered a super fast-acting insulincomposition at a dose that covers only the first phase insulin release,while Type 1 diabetics can be administered a super fast-acting insulincomposition at a dose that covers all phases of insulin release.

Thus, another use of the super fast-acting insulin compositions providedherein is to reduce the side-effects of weight gain and obesityassociated with fast-acting insulin therapy. The magnitude of this sideeffect is about or is proportional to the dose of insulin administered.As discussed above, super fast-acting insulin compositions can provideequivalent glycemic control from lower doses of fast-acting insulin asthe corresponding fast-acting compositions through, for example, acombination of the greater bioavailability and the greater fraction ofcumulative systemic insulin exposure over the first 0.25, 0.5, 0.75, 1,1.5 or 2 hours following administration. Although Type 1 and Type 2diabetic patients can experience weight gain as a result of insulintherapy, patients with Type 2 diabetes are at particular risk of weightgain, leading to obesity. Type 2 diabetics lack the first-phase insulinrelease, but still slowly release insulin over time. As a result, in theearly stages of the disease, the Type 2 diabetics' endogenous insulinlevels are too low at the initiation of a meal and too high after mealdigestion. In the absence of the first-phase insulin release, the liverdoes not receive the signal to stop making glucose. The liver continuesto produce glucose at a time when the body begins to produce new glucosethrough the digestion of the meal, resulting in hyperglycemia. Betweentwo and three hours after a meal, an untreated diabetic's blood glucosebecomes so elevated that the pancreas receives a signal to secrete alarge amount of insulin. In a patient with early Type 2 diabetes, thepancreas can still respond and secretes this large amount of insulin.This occurs at the time when digestion is almost over and blood glucoselevels should begin to fall. This large amount of insulin has twodetrimental effects. First, it puts an undue demand on an alreadycompromised pancreas, which can lead to its more rapid deterioration andeventually render the pancreas unable to produce insulin. Second, toomuch insulin after digestion can contribute to weight gain, which canfurther exacerbate the disease condition. When patients with Type 2diabetes are administered a fast-acting insulin to control postprandialhyperglycemia, as discussed above, an excess of insulin can remainfollowing digestion. Thus, Type 2 diabetic patients receiving insulintherapy can have too much insulin after digestion, which can lead tohypoglycemia and resultant weight gain. Administration of the super-fastacting insulin compositions provided herein to control postprandialhyperglycemia reduces the risk of weight gain and obesity in diabeticpatients. The super-fast acting insulin compositions can contain thelower doses of fast-acting insulin.

To achieve glycemic control, the fast-acting insulin in superfast-acting insulin compositions could be administered at 20%, 30%, 40%,50%, 60%, 70%, 80% or 90% of the level that the fast-acting insulinwould have to be administered if the hyaluronan degrading enzyme werenot present. Thus, for example, the amount of fast-acting insulinadministered in a super fast-acting composition is typically, or isabout, 0.05 U/kg, 0.06 U/kg, 0.07 U/kg, 0.08 U/kg, 0.09 U/kg, 1.0 U/kg,1.1 U/kg, 1.2 U/kg, 1.3 U/kg, 1.4 U/kg, 1.5 U/kg, 1.6 U/kg, 1.7 U/kg,1.8 U/kg, 1.9 U/kg, or 2.0 U/kg. By virtue of lower doses, the durationof action of such insulins can be lessened to minimize the potential forlate hypoglycemia that occurs due to the elevated plasma insulinconcentration that extends over several hours. Thus, a faster onset ofaction of the super fast-acting insulin composition, which more closelymimics the endogenous insulin spike of the first phase prandial insulinrelease, is expected to provide clinical benefit with regard to betterglycemic control and less weight gain in patients with diabetesmellitus.

Further, by affording an increased rate of absorption, super fast-actinginsulin compositions as described herein can provide a shorter feedbackcycle between the effect of administered insulin and effect on observedglucose levels than the corresponding fast-acting insulin compositions,and therefore are better able to mimic the natural regulation ofpostprandial glucose levels. Hence, the modified pharmacokinetics of asuper fast-acting insulin composition also benefits the performance ofthe existing ‘insulin pump’ and continuous glucose monitoring (GCM)technology. By shortening the time between a postprandial insulin bolusinjection and a systemic glycemic response, tighter control of glucoselevels from repeated smaller subcutaneous injections of insulin with GCMcould ‘close the loop’ on a combined insulin pump/glucose monitoringdevice (i.e. closed loop system or artificial pancreas).

The super fast-acting insulin compositions, whether provided as a singlemixture, or as separate preparations, of fast-acting insulin andhyaluronan degrading enzyme can contain additional ingredients toprovide desired physical or chemical properties. For example, aninjectable solution can contain one or more tonicity modifiers toprovide an approximately isotonic solution, and an aqueous solventtitrated to neutral pH with an acid or base and possibly with a pHbuffering component. Fast-acting insulin formulations often include Znand a phenolic antimicrobial preservative such as m-cresol tostructurally stabilize them in a more stable hexameric state. Metalchelators, such as EDTA, can be used to adjust the rate of dissociationof these hexamers, and other divalent metals such as calcium can bepresent to buffer the chelating capacity. Hyaluronan degrading enzymesoften require additional components to provide physical and chemicalstability, including but not limited to surfactants, oxygen scavengers,salts, amino acids and polyalcohols.

Super fast-acting insulin compositions can be presented as a kit of twoseparate containers, one containing a fast-acting insulin compositionand another containing hyaluronan degrading enzyme composition, forsequential (in any order) or concurrent coadministration; or as a kitcontaining a single container containing a mixture of a fast-actinginsulin composition and a hyaluronan degrading enzyme composition. Ifthe fast-acting insulin and the hyaluronan degrading enzyme arecoadministered, said coadministration can be sequential in any order(for example the hyaluronan degrading enzyme is administered prior tothe fast-acting insulin whereby the hyaluronan degrading enzyme degradesthe hyaluronan at the injection site prior to administration of thefast-acting insulin); or the coadministration of the fast-acting insulinand the hyaluronan degrading enzyme can be concurrent. The fast-actinginsulin composition and the hyaluronan degrading enzyme compositions canbe formulated (together or separately) as a solid for injection afterreconstitution with an appropriate diluent, as injectable solutions, oras injectable suspensions.

The following sections describe exemplary fast-acting insulins andsoluble hyaluronan degrading enzymes used in the super fast-actinginsulin compositions provided herein, methods of making them, and usingthem to treat diseases and conditions for which current fast-actinginsulins are used.

C. INSULIN POLYPEPTIDES AND FORMULATIONS

Insulin is a polypeptide composed of 51 amino acid residues that is 5808daltons in molecular weight. It is produced in the beta-cell islets ofLangerhans in the pancreas. An exemplary human insulin is translated asa 110 amino acid precursor polypeptide, preproinsulin (SEQ ID NO:101),containing a 24 amino acid signal peptide to ER, the signal sequence iscleaved, resulting in proinsulin (SEQ ID NO:102). The proinsulinmolecule is subsequently converted into a mature insulin by actions ofproteolytic enzymes, known as prohormone convertases (PC1 and PC2) andby actions of the exoprotease carboxypeptidase E. This results inremoval of 4 basic amino acid residues and the remaining 31 amino acidC-peptide or connecting chain (corresponding to amino acid residues 57to 87 of the preproinsulin polypeptide set forth in SEQ ID NO:101) Theresulting insulin contains a 21 amino acid A-chain (corresponding toamino acid residues 66 to 86 of the proinsulin polypeptide set forth inSEQ ID NO:102) and a 30 amino acid B-chain (corresponding to amino acidresidues 1 to 30 of the proinsulin polypeptide set forth in SEQ IDNO:102), which are cross-linked by disulfide bonds. Typically, matureinsulin contains three disulfide bridges: one between position 7 of theA-chain and position 7 of the B-chain, a second between position 20 ofthe A-chain and position 19 of the B-chain, and a third betweenpositions 6 and 11 of the A-chain. The sequence of the A chain of amature insulin is set forth in SEQ ID NO:103 and the sequence of theB-chain is set forth in SEQ ID NO:104.

Reference to insulin includes preproinsulin, proinsulin and insulinpolypeptides in single-chain or two-chain forms, truncated forms thereofthat have activity, and includes allelic and species variants, variantsencoded by splice variants and other variants, such as insulin analogsor other derivatized forms, including polypeptides that have at least40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more sequence identity to the precursor polypeptide setforth in SEQ ID NO:101 or the mature form thereof, so long as theinsulin binds to the human insulin receptor to initiate a signalingcascade that results in an increase of glucose uptake and storage and/ora decrease of endogenous glucose production. For example, insulinsinclude species variants of insulin. These include, but are not limitedto, insulins derived from bovine (set forth in SEQ ID NO:133) andporcine (SEQ ID NO:123). Bovine insulin differs from human insulin atamino acids 8 and 10 of the A chain, and amino acid 30 of the B chain.Porcine insulin only differs from human insulin at amino acid 30 in theB chain where, like the bovine sequence, there is an alaninesubstitution in place of threonine. Other exemplary species variants ofinsulin are set forth in any of SEQ ID NOS: 105-146. Also included amongvariants of insulin are insulin analogs that contain one or more aminoacid modifications compared to a human insulin set forth in SEQ ID NO:103 and 104 (A and B chains). Exemplary insulin analogs (A and Bchains), including fast-acting and longer-acting analog forms, are setforth in SEQ ID NOS:147-165, 182-184). For example, insulin analogsinclude, but are not limited to, glulisine (LysB3, GluB29; set forth inSEQ ID NO:103 (A-chain) and SEQ ID NO:149 (B-chain)), HMR-1 153 (LysB3,IleB28; set forth in SEQ ID NO:103 (A-chain) and SEQ ID NO:182(B-chain)), HMR-1423 (GlyA21, HisB31, HisB32; set forth in SEQ ID NO:183(A-chain) and SEQ ID NO:184 (B-chain)), insulin aspart (AspB28; setforth in SEQ ID NO:103 (A-chain) and SEQ ID NO:147 (B-chain)), andinsulin lispro (LysB28, ProB29; set forth in SEQ ID NO:103 (A-chain) andSEQ ID NO:148 (B-chain)). In every instance above, the nomenclature ofthe analogs is based on a description of the amino acid substitution atspecific positions on the A or B chain of insulin, numbered from theN-terminus of the chain, in which the remainder of the sequence is thatof natural human insulin.

Any of the above insulin polypeptides include those that are produced bythe pancreas from any species, such as a human, and also includeinsulins that are produced synthetically or using recombinanttechniques. For example, as described elsewhere herein, insulin can beproduced biosynthetically by expressing synthetic genes for A and Bchains of insulin, by expressing the entire proinsulin and exposing itto the appropriate enzymatic and chemical methods to generate a matureinsulin, or by expressing A and B chains connected by a linker peptide(see e.g., DeFelippis et al. (2002) Insulin Chemistry andPharmacokinetics. In Ellenberg and Rifkin's Diabetes Mellitus (pp.481-500) McGraw-Hill Professional).

Insulins also include monomeric and oligomeric forms, such as hexamericforms. Insulin can exist as a monomer as it circulates in the plasma,and it also binds to its receptor while in a monomeric form. Insulin,however, has a propensity to self-associate into dimers, and in thepresence of metal ions such as Zn²⁺ can readily associate into higherorder structures such as hexamers. There are two symmetrical highaffinity binding sites for Zn²⁺, although other weaker zinc-bindingsites also have been reported (see e.g., DeFelippis et al. (2002)Insulin Chemistry and Pharmacokinetics. In Ellenberg and Rifkin'sDiabetes Mellitus (pp. 481-500) McGraw-Hill Professional).Self-association is important for the stability of the molecule toprevent chemical degradation and physical denaturation. Thus, in storagevesicles in pancreatic beta-cells, insulin exists as a hexamer. Uponrelease into the extracellular space, however, it is believed that theinsulin hexamers can experience a change in pH to more neutralconditions and the zinc ion-containing hexamers are diluted, whichdestabilizes the hexamer. There may be other reasons contributing to thedestabilization of the insulin hexamer in the extracellular space.Insulin is thus predominantly found in the blood as a monomer. To takeadvantage of the stabilizing effects, most commercial formulations ofinsulin contain zinc ions in sufficient amounts to promoteself-association into hexamers. The hexameric structure, however, slowsdown the absorption rate of these formulations upon subcutaneousadministration.

As discussed in Section B, insulin is used as a therapeutic for glycemiccontrol, such as in diabetic patients. There are various types ofinsulin formulations that exist, depending on whether the insulin isbeing administered to control glucose for basal therapy, for prandialtherapy, or for a combination thereof. Insulin formulations can beprovided solely as fast-acting formulations, solely as basal-actingformulations (i.e., intermediate-acting and/or long-acting forms), or asmixtures thereof (see e.g., Table 2). Typically, mixtures contain afast-acting and an intermediate- or long-acting insulin. For example,fast-acting insulins can be combined with an NPH insulin (an exemplaryintermediate-acting insulin as discussed below) in various mixtureratios including 10:90, 20:80, 30:70, 40:60, and 50:50. Such premixedpreparations can reduce the number of daily insulin injections byconveniently providing both meal-related and basal insulin requirementsin a single formulation. Accordingly, the super fast-acting insulincomposition formulations described herein include those that optionallycan provide a basal-acting insulin.

Generally, any preparation of insulin includes an insulin polypeptide orvariant (i.e. analog) thereof, and differ only in the other substancesthat make up the formulation. Hence, it is the specifics of theformulation that can influence the duration of action of differentinsulin types. Examples of substances included in insulin preparations,include, but are not limited to, stabilization agents such as zinc, pHbuffer, a tonicity modifier such as glycerin; apreservative/anti-microbial agent such as m-cresol; and protamine orother precipitation or controlled release agent. Further, as providedherein, insulin preparations also can be prepared containing calcium anda metal chelator such as EDTA or EGTA. Any one or more of the abovesubstances can be added to an insulin polypeptide, such as in a superfast-acting insulin composition. The specific components added, andtheir amounts, influence the type of insulin, its duration of action,its absorption and bioavailability and hence, its application.

For example, most insulin preparations contain a metal ion, such aszinc, in the formulation, which stabilizes the insulin by promotingself-association of the molecule. Self-association into hexameric formscan affect the absorption of insulin upon administration. Hence, theratio of such stabilizing agents, and the addition of EDTA or EGTA toinsulin, permits further modulation and control of the absorption andbioavailability of insulin, for example, by influencing the prevalenceof higher order structure present in the polypeptide. Generally, regularinsulin preparations that are fast-acting contain zinc in an amount thatis or is about 0.01-0.04 mg/100 Units. Chemical studies have revealedthat the solubility of insulin is largely determined by the zinc contentand the nature of the buffer in which it is suspended. Hence, somelonger-acting basal insulin formulations are prepared by precipitatinginsulin from an acetate buffer (instead of phosphate) by the addition ofzinc. Large crystals of insulin with high zinc content, when collectedand resuspended in a solution of sodium acetate-sodium chloride (pH 7.2to 7.5), are slowly absorbed after subcutaneous injection and exert anaction of long duration. This crystal preparation is named extendedinsulin zinc suspension (ultralente insulin). Other zinc-containinginsulin preparations include, for example, semilente insulins (promptinsulin zinc suspensions) and lente insulins (insulin zinc suspensions),which differ predominantly in the zinc concentration used.Zinc-containing insulin preparations also include those that aremodified by protamine, such as NPH insulin.

In another example, a precipitation agent, such as protamine, can beadded to an insulin polypeptide to generate a microcrystallinesuspension. Typically, crystalline insulins have a prolonged duration ofaction compared to insulins that do not exist in crystalline form. Aprotamine zinc insulin, when injected subcutaneously in an aqueoussuspension, dissolves only slowly at the site of deposition, and theinsulin is absorbed at a retarded rate. Protamine zinc suspensioninsulin has largely been replaced by isophane insulin suspension, alsoknown as NPH insulin. It is a modified protamine zinc insulin suspensionthat is crystalline. The concentrations of insulin, protamine, and zincare so arranged that the preparation has an onset and a duration ofaction intermediate between those of regular insulin and protamine zincinsulin suspension.

Further, pH differences in the preparations also influence the type andproperty of insulin. The original regular insulin preparations wereprepared at a pH of 2.8 to 3.5, otherwise they would form particles athigher pH ranges. Highly purified insulin preparations, however, can beprepared at a range of pH values. Also, buffering the insulinpreparation allows insulin to be prepared in a solution over a widerrange of pH. Typically, an insulin that is prepared at neutral pH has agreater stability then those prepared at acidic pH. Thus, most insulinsare formulated at neutral pH. An exception is insulin glargine, which isprovided as a commercial formulation at pH 4.0. By virtue of theaddition of two arginines to the C-terminus of the B-chain, theisoelectric point of the glargine insulin is shifted making it moresoluble at an acidic pH. An additional amino acid change exists in the Achain (N21G) to prevent deamidation and dimerization resulting from anacid-sensitive asparagine. The sequence of the A chain of glargineinsulin is set forth in SEQ ID NO:150 and the B-chain is set forth inSEQ ID NO:151. Since exposure to physiologic pH occurs uponadministration, microprecipitates are formed, which make glarginesimilar to a crystalline, long-acting insulin.

Table 2 below summarizes various types of insulin, their onset of actionand their application.

TABLE 2 Types of Insulins Type Brand name Onset Peak DurationApplication Fast-acting: Lispro (e.g. 5-15 minutes 45-90 minutes  3-4hours Post-prandial Insulin Humalog ®); glucose control analogs Aspart(e.g., NovoLog ®); Glulisine Fast-acting: Regular 30 minutes-1 hour  2-5hours  5-8 hours Post-prandial Regular Insulin (e.g., glucose controlinsulin Humulin ® R; Novolin ® R; Velosulin ® Human) Intermediate-Lente ® (e.g.,  1-3 hours  6-12 hours 20-24 hours Basal insulin ActingHumulin ® L, supplementation Novolin ® L); NPH (e.g., Humulin ® N,Novolin ® N); Long-lasting Ultralente  4-6 hours 18-28 hours   28 hoursBasal insulin (e.g. supplementation Humulin ® U); glargine; detemir (ananalog) Mixtures Humulin ® Varies Varies Varies 50/50; Humulin ® 70/30;Novolin ® 70/30; Humalog ® Mix 75/25

The most commonly used insulins are fast-acting insulins, which includeregular insulin (i.e. native or wildtype insulin, including allelic andspecies variants thereof) and fast-acting insulin analogs. For purposesherein, reference to insulin is a fast-acting insulin, unlessspecifically noted otherwise.

Fast-Acting Insulin

Provided herein are super fast-acting insulin compositions that containa fast-acting insulin and a soluble hyaluronan degrading enzyme.Generally, these super fast-acting insulin compositions are absorbedfollowing subcutaneous administration and are detectable and have anonset of action in the blood within 30 minutes or less. Fast-actinginsulins that can be used to obtain a super fast-acting insulincomposition as described herein include regular insulin, which is thewild-type or native insulin. Fast-acting insulins also include insulinanalogs. By virtue of their fast absorption rate compared tobasal-acting insulins, fast-acting insulins are used predominantly forpost-prandial control purposes. Exemplary fast-acting insulins are setforth in Table 3 below. Fast-acting insulins also include any known inthe art, such as, but not limited to, any insulin preparations anddevices disclosed in U.S. Pat. No. 7,279,457 and US Patent Publications20070235365, 20080039368, 20080039365, 20070086952, 20070244467, and20070191757. Any fast-acting insulin can be rendered super fast-actingby co-formulation and/or co-administration with a hyaluronan degradingenzyme. A super fast-acting insulin composition formulation also canfurther include a mixture of a fast-acting insulin with an intermediateor long-acting insulin, in addition to a hyaluronan degrading enzyme.

TABLE 3 FAST-ACTING INSULINS A-chain B-chain (SEQ ID (SEQ ID CommercialName Species NO) NO) Name Regular Human SEQ ID SEQ ID e.g. Insulin NO:103 NO: 104 Humulin ®; Novolin ® R; Velosulin ® Regular Porcine 88-108of 25-54 of Iletin II ®; Insulin SEQ ID SEQ ID NO: 123 NO: 123 AspartHuman SEQ ID SEQ ID Novolog ® Insulin analog NO: 103 NO: 147 LisproHuman SEQ ID SEQ ID Humalog ® Insulin analog NO: 103 NO: 148 GlulisineHuman SEQ ID SEQ ID Apidra ® Insulin analog NO: 103 NO: 149

a. Regular Insulin

Regular insulins include formulations that include the native orwildtype insulin polypeptide. These include human insulin, as well asinsulins from bovine, porcine and other species. Such insulins can beprepared at an acidic pH (e.g., 2.5-3.5) or can be prepared at a neutralpH (e.g., 7.0-7.8). Regular insulins also include those that containzinc. Typically, the zinc content in regular insulin preparations rangesfrom at or about 0.01-0.04 mg/100 Units. Regular human insulins aremarketed as Humulin® R, Novolin® R and Velosulin®. Porcine insulin wasmarketed as Iletin II®. Generally, regular insulin has an onset ofaction of 30 minutes after subcutaneous administration. Maximal plasmalevels are seen in 1-3 hours and the duration of intensity increaseswith dosage. The plasma half-life following subcutaneous administrationis about 1.5 hours.

b. Fast-Acting Analogs

Fast-Acting insulin analogs are modified forms of insulin that typicallycontain one or more amino acid changes. The analogs are designed toreduce the self-association of the insulin molecule for the purpose ofincreasing the absorption rate and onset of action as compared toregular insulin. Generally, such analogs are formulated in the presenceof zinc, and thus exist as stable zinc hexamers. Due to themodification, however, they have a quicker dissociation from thehexameric state after subcutaneous administration compared to regularinsulin.

i. Insulin Lispro

Human insulin Lispro is an insulin polypeptide formulation containingamino acid changes at position 28 and 29 of the B-chain such that thePro-Lys at this position in wild-type insulin B-chain set forth in SEQID NO:104 is inverted to Lys-Pro. The sequence of insulin lispro is setforth in SEQ ID NO:103 (A-chain) and SEQ ID NO: 148 (B-chain). It ismarketed under the name Humalog®. The result of the inversion of thesetwo amino acids is a polypeptide with a decreased propensity toself-associate, which allows for a more rapid onset of action.Specifically, the sequence inversion in the B-chain results in theelimination of two hydrophobic interactions and weakening of twobeta-pleated sheet hydrogen bonds that stabilize the dimer (see e.g.,DeFelippis et al. (2002) Insulin Chemistry and Pharmacokinetics. InEllenberg and Rifkin's Diabetes Mellitus (pp. 481-500) McGraw-HillProfessional). The polypeptide self-associates and forms hexamers as aresult of excipients provided in the formulation, such as antimicrobialagents (e.g. m-cresol) and zinc for stabilization. Nevertheless, due tothe amino acid modification, insulin lispro is more rapidly acting thenregular insulin.

ii. Insulin Aspart

Human insulin aspart is an insulin polypeptide formulation containing anamino acid substitution at position 28 of the B-chain of human insulinset forth in SEQ ID NO:104 from a proline to an aspartic acid. Thesequence of insulin aspart is set forth in SEQ ID NO:103 (A-chain) andSEQ ID NO:147 (B-chain). It is marketed under the name Novolog®. Themodification in insulin aspart confers a negatively-charged side-chaincarboxyl group to create charge repulsion and destabilize themonomer-monomer interaction. Further, the removal of the prolineeliminates a key hydrophobic interaction between monomers (see e.g.,DeFelippis et al. (2002) Insulin Chemistry and Pharmacokinetics. InEllenberg and Rifkin's Diabetes Mellitus (pp. 481-500) McGraw-HillProfessional). The analog exists largely as a monomer, and is less proneto aggregate compared to other fast-acting analogs such as lispro.Generally, insulin aspart and insulin lispro are similar in theirrespective pharmacokinetic and phamacodynamic properties.

iii. Insulin Glulisine

Human insulin glulisine is an insulin polypeptide formulation containingan amino acid substitution in the B-chain at position B3 from asparagineto lysine and at amino acid B29 from lysine to glutamic acid compared tothe sequence of the B-chain of human insulin set forth in SEQ ID NO:104.The sequence of insulin glulisine is set forth in SEQ ID NO:103(A-chain) and SEQ ID NO:149 (B-chain). It is marketed under the nameApidra®. The modifications render the polypeptide molecule less prone toself-association compared to human insulin. Unlike other insulinanalogs, the polypeptide is commercially formulated in the absence ofthe hexamer-promoting zinc (Becker et al. (2008) ClinicalPharmacokinetics, 47:7-20). Hence, insulin glulisine has a more rapidrate of onset than insulin lispro and insulin aspart.

D. HYALURONAN DEGRADING ENZYMES

Provided herein are super fast-acting insulin compositions andcombinations resulting from combination of a fast-acting insulin and ahyaluronan (hyaluronic acid) degrading enzyme, and methods of using suchcompositions and combinations for the treatment of insulin-mediateddiseases and conditions. Hyaluronan degrading enzymes include any enzymethat degrades hyaluronan. Exemplary hyaluronan degrading enzymesinclude, but are not limited to hyaluronidases and particularchondroitinases and lyases that have the ability to cleave hyaluronan.Where the methods and uses provided herein describe the use of ahyaluronidase with insulin, accordingly any hyaluronan degrading enzymecan be used. Exemplary of hyaluronan degrading enzymes in thecompositions, combinations and methods provided herein are solublehyaluronan degrading enzymes. By virtue of the ability of hyaluronandegrading enzymes, such as a hyaluronidase, to break down hyaluronicacid in the extracellular matrix, such enzymes, facilitateadministration of therapeutic agents. For example, the absorption anddispersion of therapeutics that are co-administered with a hyaluronandegrading enzyme such as by subcutaneous administration, are increased.

Hyaluronan, also called hyaluronic acid or hyaluronate, is anon-sulfated glycosaminoglycan that is widely distributed throughoutconnective, epithelial, and neural tissues. Hyaluronan is an essentialcomponent of the extracellular matrix and a major constituent of theinterstitial barrier. By catalyzing the hydrolysis of hyaluronan,hyaluronan degrading enzymes lower the viscosity of hyaluronan, therebyincreasing tissue permeability and increasing the absorption rate offluids administered parenterally. As such, hyaluronan degrading enzymes,such as hyaluronidases, have been used, for example, as spreading ordispersing agents in conjunction with other agents, drugs and proteinsto enhance their dispersion and delivery.

Hyaluronan degrading enzymes act to degrade hyaluronan by cleavinghyaluronan polymers, which are composed of repeating disaccharidesunits, D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc),linked together via alternating β-1→4 and β-1→3 glycosidic bonds.Hyaluronan chains can reach about 25,000 disaccharide repeats or more inlength and polymers of hyaluronan can range in size from about 5,000 to20,000,000 Da in vivo. Accordingly, hyaluronan degrading enzymes for theuses and methods provided include any enzyme having the ability tocatalyze the cleavage of a hyaluronan disaccharide chain or polymer. Insome examples the hyaluronan degrading enzyme cleaves the β-1→4glycosidic bond in the hyaluronan chain or polymer. In other examples,the hyaluronan degrading enzyme catalyze the cleavage of the β-1→3glycosidic bond in the hyaluronan chain or polymer.

As described below, hyaluronan-degrading enzymes exist in membrane-boundor soluble form. For purposes herein, soluble hyaluronan-degradingenzymes are provided for use in the methods, uses, compositions orcombinations herein. Thus, where hyaluronan-degrading enzymes include aglycosylphosphatidylinositol (GPI) anchor and/or are otherwisemembrane-anchored or insoluble, hyaluronan-degrading enzymes areprovided herein in soluble form. Thus, hyaluronan-degrading enzymesinclude truncated variants, e.g. truncated to remove all or a portion ofa GPI anchor. Hyaluronan-degrading enzymes provide herein also includeallelic or species variants or other variants, of a solublehyaluronan-degrading enzyme. For example, hyaluronan degrading enzymescan contain one or more variations in its primary sequence, such asamino acid substitutions, additions and/or deletions. A variant of ahyaluronan-degrading enzyme generally exhibits at least or about 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity compared to the hyaluronan-degrading enzyme notcontaining the variation. Any variation can be included in thehyaluronan degrading enzyme for the purposes herein provided the enzymeretains hyaluronidase activity, such as at least or about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or more of the activity of a hyaluronan degrading enzyme notcontaining the variation (as measured by in vitro and/or in vivo assayswell known in the art and described herein).

1. Hyaluronidases

Hyaluronidases are members of a large family of hyaluronan degradingenzymes. There are three general classes of hyaluronidases:mammalian-type hyaluronidases, bacterial hyaluronidases andhyaluronidases from leeches, other parasites and crustaceans. Suchenzymes can be used in the compositions, combinations and methodsprovided.

a. Mammalian-Type Hyaluronidases Mammalian-type hyaluronidases (EC3.2.1.35) are endo-β-N-acetyl-hexosaminidases that hydrolyze the β-1-4glycosidic bond of hyaluronan into various oligosaccharide lengths suchas tetrasaccharides and hexasaccharides. These enzymes have bothhydrolytic and transglycosidase activities, and can degrade hyaluronanand chondroitin sulfates (CS), generally C4-S and C6-S. Hyaluronidasesof this type include, but are not limited to, hyaluronidases from cows(bovine) (SEQ ID NOS:10, 11 and 64 and BH55 (U.S. Pat. Nos. 5,747,027and 5,827,721)), sheep (ovis aries) (SEQ ID NO: 26, 27, 63 and 65),yellow jacket wasp (SEQ ID NOS:12 and 13), honey bee (SEQ ID NO:14),white-face hornet (SEQ ID NO:15), paper wasp (SEQ ID NO:16), mouse (SEQID NOS:17-19, 32), pig (SEQ ID NOS:20-21), rat (SEQ ID NOS:22-24, 31),rabbit (SEQ ID NO:25), orangutan (SEQ ID NO:28), cynomolgus monkey (SEQID NO:29), guinea pig (SEQ ID NO:30), and human hyaluronidases.Exemplary of hyaluronidases in the compositions, combinations andmethods provided herein are soluble hyaluronidases

Mammalian hyaluronidases can be further subdivided into those that areneutral active, predominantly found in testes extracts, and acid active,predominantly found in organs such as the liver. Exemplary neutralactive hyaluronidases include PH20, including but not limited to, PH20derived from different species such as ovine (SEQ ID NO:27), bovine (SEQID NO:11) and human (SEQ ID NO:1). Human PH20 (also known as SPAM1 orsperm surface protein PH20), is generally attached to the plasmamembrane via a glycosylphosphatidyl inositol (GPI) anchor. It isnaturally involved in sperm-egg adhesion and aids penetration by spermof the layer of cumulus cells by digesting hyaluronic acid.

Besides human PH20 (also termed SPAM1), five hyaluronidase-like geneshave been identified in the human genome, HYAL1, HYAL2, HYAL3, HYAL4 andHYALP1. HYALP1 is a pseudogene, and HYAL3 (SEQ ID NO:38) has not beenshown to possess enzyme activity toward any known substrates. HYAL4(precursor polypeptide set forth in SEQ ID NO:39) is a chondroitinaseand exhibits little activity towards hyaluronan. HYAL1 (precursorpolypeptide set forth in SEQ ID NO:36) is the prototypical acid-activeenzyme and PH20 (precursor polypeptide set forth in SEQ ID NO:1) is theprototypical neutral-active enzyme. Acid-active hyaluronidases, such asHYAL1 and HYAL2 (precursor polypeptide set forth in SEQ ID NO:37)generally lack catalytic activity at neutral pH (i.e. pH 7). Forexample, HYAL1 has little catalytic activity in vitro over pH 4.5 (Frostet al. (1997) Anal. Biochem. 251:263-269). HYAL2 is an acid-activeenzyme with a very low specific activity in vitro. Thehyaluronidase-like enzymes also can be characterized by those which aregenerally attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor such as human HYAL2 and human PH20(Danilkovitch-Miagkova et al. (2003) Proc Natl Acad Sci USA100(8):4580-5), and those which are generally soluble such as humanHYAL1 (Frost et al. (1997) Biochem Biophys Res Commun. 236(1):10-5).

PH20

PH20, like other mammalian hyaluronidases, is anendo-β-N-acetyl-hexosaminidase that hydrolyzes the β1→4 glycosidic bondof hyaluronic acid into various oligosaccharide lengths such astetrasaccharides and hexasaccharides. They have both hydrolytic andtransglycosidase activities and can degrade hyaluronic acid andchondroitin sulfates, such as C4-S and C6-S. PH20 is naturally involvedin sperm-egg adhesion and aids penetration by sperm of the layer ofcumulus cells by digesting hyaluronic acid. PH20 is located on the spermsurface, and in the lysosome-derived acrosome, where it is bound to theinner acrosomal membrane. Plasma membrane PH20 has hyaluronidaseactivity only at neutral pH, while inner acrosomal membrane PH20 hasactivity at both neutral and acid pH. In addition to being ahyaluronidase, PH20 also appears to be a receptor for HA-induced cellsignaling, and a receptor for the zona pellucida surrounding the oocyte.

Exemplary PH20 proteins include, but are not limited to, human(precursor polypeptide set forth in SEQ ID NO:1, mature polypeptide setforth in SEQ ID NO: 2), chimpanzee (SEQ ID NO:185), Rhesus monkey (SEQID NO:186) bovine (SEQ ID NOS: 11 and 64), rabbit (SEQ ID NO: 25), ovinePH20 (SEQ ID NOS: 27, 63 and 65), Cynomolgus monkey (SEQ ID NO: 29),guinea pig (SEQ ID NO: 30), rat (SEQ ID NO: 31) and mouse (SEQ ID NO:32) PH20 polypeptides. Bovine PH20 is a 553 amino acid precursorpolypeptide (SEQ ID NO:11). Alignment of bovine PH20 with the human PH20shows only weak homology, with multiple gaps existing from amino acid470 through to the respective carboxy termini due to the absence of aGPI anchor in the bovine polypeptide (see e.g., Frost GI (2007) ExpertOpin. Drug. Deliv. 4: 427-440). In fact, clear GPI anchors are notpredicted in many other PH20 species besides humans. Thus, PH20polypeptides produced from ovine and bovine naturally exist as solubleforms. Though bovine PH20 exists very loosely attached to the plasmamembrane, it is not anchored via a phospholipase sensitive anchor(Lalancette et al. (2001) Biol Reprod. 65(2):628-36). This uniquefeature of bovine hyaluronidase has permitted the use of the solublebovine testes hyaluronidase enzyme as an extract for clinical use(Wydase®, Hyalase®).

The human PH20 mRNA transcript is normally translated to generate a 509amino acid precursor polypeptide (SEQ ID NO:1; and replicated below)containing a 35 amino acid signal sequence at the N-terminus (amino acidresidue positions 1-35) and a 19 amino acid glycosylphosphatidylinositol(GPI) anchor attachment signal sequence at the C-terminus (amino acidresidue positions 491-509). The mature PH20 is, therefore, a 474 aminoacid polypeptide set forth in SEQ ID NO:2. Following transport of theprecursor polypeptide to the ER and removal of the signal peptide, theC-terminal GPI-attachment signal peptide is cleaved to facilitatecovalent attachment of a GPI anchor to the newly-formed C-terminal aminoacid at the amino acid position corresponding to position 490 of theprecursor polypeptide set forth in SEQ ID NO:1. Thus, a 474 amino acidGPI-anchored mature polypeptide with an amino acid sequence set forth inSEQ ID NO:2 is produced.

Amino acid sequence of the human PH20 precursor polypeptide (SEQ IDNO:1; 509 amino acids):

MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL

Human PH20 exhibits hyaluronidase activity at both neutral and acid pH.In one aspect, human PH20 is the prototypical neutral-activehyaluronidase that is generally locked to the plasma membrane via a GPIanchor. In another aspect, PH20 is expressed on the inner acrosomalmembrane where it has hyaluronidase activity at both neutral and acidpH. It appears that PH20 contains two catalytic sites at distinctregions of the polypeptide: the Peptide 1 and Peptide 3 regions (Cherret al., (2001) Matrix Biology 20:515-525). Evidence suggests that thePeptide 1 region of PH20, which corresponds to amino acid positions107-137 of the mature polypeptide set forth in SEQ ID NO:2 and positions142-172 of the precursor polypeptide set forth in SEQ ID NO:1, isrequired for enzyme activity at neutral pH. Amino acids at positions 111and 113 (corresponding to the mature PH20 polypeptide set forth in SEQID NO:2) within this region appear to be important for activity, asmutagenesis by amino acid replacement results in PH20 polypeptides with3% hyaluronidase activity or undetectable hyaluronidase activity,respectively, compared to the wild-type PH20 (Arming et al., (1997) Eur.J. Biochem. 247:810-814).

The Peptide 3 region, which corresponds to amino acid positions 242-262of the mature polypeptide set forth in SEQ ID NO:2, and positions277-297 of the precursor polypeptide set forth in SEQ ID NO: 1, appearsto be important for enzyme activity at acidic pH. Within this region,amino acids at positions 249 and 252 of the mature PH20 polypeptideappear to be essential for activity, and mutagenesis of either oneresults in a polypeptide essentially devoid of activity (Arming et al.,(1997) Eur. J. Biochem. 247:810-814).

In addition to the catalytic sites, PH20 also contains ahyaluronan-binding site. Experimental evidence suggest that this site islocated in the Peptide 2 region, which corresponds to amino acidpositions 205-235 of the precursor polypeptide set forth in SEQ ID NO: 1and positions 170-200 of the mature polypeptide set forth in SEQ IDNO:2. This region is highly conserved among hyaluronidases and issimilar to the heparin binding motif. Mutation of the arginine residueat position 176 (corresponding to the mature PH20 polypeptide set forthin SEQ ID NO:2) to a glycine results in a polypeptide with only about 1%of the hyaluronidase activity of the wild type polypeptide (Arming etal., (1997) Eur. J. Biochem. 247:810-814).

There are seven potential N-linked glycosylation sites in human PH20 atN82, N166, N235, N254, N368, N393, N490 of the polypeptide exemplifiedin SEQ ID NO: 1. Because amino acids 36 to 464 of SEQ ID NO:1 appears tocontain the minimally active human PH20 hyaluronidase domain, theN-linked glycosylation site N-490 is not required for properhyaluronidase activity. There are six disulfide bonds in human PH20. Twodisulphide bonds between the cysteine residues C60 and C351 and betweenC224 and C238 of the polypeptide exemplified in SEQ ID NO: 1(corresponding to residues C25 and C316, and C189 and C203 of the maturepolypeptide set forth in SEQ ID NO:2, respectively). A further fourdisulphide bonds are formed between the cysteine residues C376 and C387;between C381 and C435; between C437 and C443; and between C458 and C464of the polypeptide exemplified in SEQ ID NO: 1 (corresponding toresidues C341 and C352; between C346 and C400; between C402 and C408;and between C423 and C429 of the mature polypeptide set forth in SEQ IDNO:2, respectively).

b. Bacterial Hyaluronidases

Bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1) degrade hyaluronanand, to various extents, chondroitin sulfates and dermatan sulfates.Hyaluronan lyases isolated from bacteria differ from hyaluronidases(from other sources, e.g., hyaluronoglucosaminidases, EC 3.2.1.35) bytheir mode of action. They are endo-β-N-acetylhexosaminidases thatcatalyze an elimination reaction, rather than hydrolysis, of theβ1→4-glycosidic linkage between N-acetyl-beta-D-glucosamine andD-glucuronic acid residues in hyaluronan, yielding3-(4-deoxy-β-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine tetra- andhexasaccharides, and disaccharide end products. The reaction results inthe formation of oligosaccharides with unsaturated hexuronic acidresidues at their nonreducing ends.

Exemplary hyaluronidases from bacteria for use in the compositions,combinations and methods provided include, but are not limited to,hyaluronan degrading enzymes in microorganisms, including strains ofArthrobacter, Bdellovibrio, Clostridium, Micrococcus, Streptococcus,Peptococcus, Propionibacterium, Bacteroides, and Streptomyces.Particular examples of such enzymes include, but are not limited toArthrobacter sp. (strain FB24) (SEQ ID NO:67), Bdellovibriobacteriovorus (SEQ ID NO:68), Propionibacterium acnes (SEQ ID NO:69),Streptococcus agalactiae ((SEQ ID NO:70); 18RS21 (SEQ ID NO:71);serotype Ia (SEQ ID NO:72); serotype III (SEQ ID NO:73), Staphylococcusaureus (strain COL) (SEQ ID NO:74); strain MRSA252 (SEQ ID NOS:75 and76); strain MSSA476 (SEQ ID NO:77); strain NCTC 8325 (SEQ ID NO:78);strain bovine RF122 (SEQ ID NOS:79 and 80); strain USA300 (SEQ IDNO:81), Streptococcus pneumoniae ((SEQ ID NO:82); strain ATCC BAA-255/R6(SEQ ID NO:83); serotype 2, strain D39/NCTC 7466 (SEQ ID NO:84),Streptococcus pyogenes (serotype M1) (SEQ ID NO:85); serotype M2, strainMGAS10270 (SEQ ID NO:86); serotype M4, strain MGAS10750 (SEQ ID NO:87);serotype M6 (SEQ ID NO:88); serotype M12, strain MGAS2096 (SEQ ID NOS:89and 90); serotype M12, strain MGAS9429 (SEQ ID NO:91); serotype M28 (SEQID NO:92); Streptococcus suis (SEQ ID NOS:93-95); Vibrio fischeri(strain ATCC 700601/ES114 (SEQ ID NO:96)), and the Streptomyceshyaluronolyticus hyaluronidase enzyme, which is specific for hyaluronicacid and does not cleave chondroitin or chondroitin sulfate (Ohya, T.and Kaneko, Y. (1970) Biochim. Biophys. Acta 198:607).

c. Hyaluronidases from Leeches, Other Parasites and Crustaceans

Hyaluronidases from leeches, other parasites, and crustaceans (EC3.2.1.36) are endo-β-glucuronidases that generate tetra- andhexasaccharide end-products. These enzymes catalyze hydrolysis of1→3-linkages between β-D-glucuronate and N-acetyl-D-glucosamine residuesin hyaluronate. Exemplary hyaluronidases from leeches include, but arenot limited to, hyaluronidase from Hirudinidae (e.g., Hirudomedicinalis), Erpobdellidae (e.g., Nephelopsis obscura and Erpobdellapunctata), Glossiphoniidae (e.g., Desserobdella picta, Helobdellastagnalis, Glossiphonia complanata, Placobdella ornata and Theromyzonsp.) and Haemopidae (Haemopis marmorata) (Hovingh et al. (1999) CompBiochem Physiol B Biochem Mol Biol. 124(3):319-26). An exemplaryhyaluronidase from bacteria that has the same mechanism of action as theleech hyaluronidase is that from the cyanobacteria, Synechococcus sp.(strain RCC307, SEQ ID NO:97).

2. Other Hyaluronan Degrading Enzymes

In addition to the hyaluronidase family, other hyaluronan degradingenzymes can be used in conjunction with the fast-acting insulin in thecompositions, combinations and methods provided. For example, enzymes,including particular chondroitinases and lyases, that have the abilityto cleave hyaluronan can be employed. Exemplary chondroitinases that candegrade hyaluronan include, but are not limited to, chondroitin ABClyase (also known as chondroitinase ABC), chondroitin AC lyase (alsoknown as chondroitin sulfate lyase or chondroitin sulfate eliminase) andchondroitin C lyase. Methods for production and purification of suchenzymes for use in the compositions, combinations, and methods providedare known in the art (e.g., U.S. Pat. No. 6,054,569; Yamagata et al.(1968) J. Biol. Chem. 243(7):1523-1535; Yang et al. (1985) J. Biol.Chem. 160(30):1849-1857).

Chondroitin ABC lyase contains two enzymes, chondroitin-sulfate-ABCendolyase (EC 4.2.2.20) and chondroitin-sulfate-ABC exolyase (EC4.2.2.21) (Hamai et al. (1997) J Biol Chem. 272(14):9123-30), whichdegrade a variety of glycosaminoglycans of the chondroitin-sulfate- anddermatan-sulfate type. Chondroitin sulfate, chondroitin-sulfateproteoglycan and dermatan sulfate are the preferred substrates forchondroitin-sulfate-ABC endolyase, but the enzyme also can act onhyaluronan at a lower rate. Chondroitin-sulfate-ABC endolyase degrades avariety of glycosaminoglycans of the chondroitin-sulfate- anddermatan-sulfate type, producing a mixture of Δ4-unsaturatedoligosaccharides of different sizes that are ultimately degraded toΔ4-unsaturated tetra- and disaccharides. Chondroitin-sulfate-ABCexolyase has the same substrate specificity but removes disaccharideresidues from the non-reducing ends of both polymeric chondroitinsulfates and their oligosaccharide fragments produced bychondroitin-sulfate-ABC endolyase (Hamai, A. et al. (1997) J. Biol.Chem. 272:9123-9130). A exemplary chondroitin-sulfate-ABC endolyases andchondroitin-sulfate-ABC exolyases include, but are not limited to, thosefrom Proteus vulgaris and Flavobacterium heparinum (the Proteus vulgarischondroitin-sulfate-ABC endolyase is set forth in SEQ ID NO: 98 (Sato etal. (1994) Appl. Microbiol. Biotechnol. 41(1):39-46).

Chondroitin AC lyase (EC 4.2.2.5) is active on chondroitin sulfates Aand C, chondroitin and hyaluronic acid, but is not active on dermatansulfate (chondroitin sulfate B). Exemplary chondroitinase AC enzymesfrom the bacteria include, but are not limited to, those fromFlavobacterium heparinum and Victivallis vadensis, set forth in SEQ IDNOS:99 and 100, respectively, and Arthrobacter aurescens (Tkalec et al.(2000) Applied and Environmental Microbiology 66(1):29-35; Ernst et al.(1995) Critical Reviews in Biochemistry and Molecular Biology30(5):387-444).

Chondroitinase C cleaves chondroitin sulfate C producing tetrasaccharideplus an unsaturated 6-sulfated disaccharide (delta Di-6S). It alsocleaves hyaluronic acid producing unsaturated non-sulfated disaccharide(delta Di-OS). Exemplary chondroitinase C enzymes from the bacteriainclude, but are not limited to, those from Streptococcus andFlavobacterium (Hibi et al. (1989) FEMS-Microbiol-Lett. 48(2):121-4;Michelacci et al. (1976) J. Biol. Chem. 251:1154-8; Tsuda et al. (1999)Eur. J. Biochem. 262:127-133)

3. Soluble Hyaluronan Degrading Enzymes

Provided in the compositions, combinations and methods herein aresoluble hyaluronan degrading enzymes, including soluble hyaluronidases.Soluble hyaluronan degrading enzymes include any hyaluronan degradingenzymes that exist in soluble form, including, but not limited to,soluble hyaluronidases, including non-human soluble hyaluronidases,including non-human animal soluble hyaluronidases, bacterial solublehyaluronidases and human hyaluronidases, Hyal1, bovine PH20 and ovinePH20, allelic variants thereof and other variants thereof. For example,included among soluble hyaluronan degrading enzymes are any hyaluronandegrading enzymes that have been modified to be soluble, including anydescribed in U.S. Provisional Application Ser. No. 61/201,384(incorporated by reference in its entirety). For example, hyaluronandegrading enzymes that contain a GPI anchor can be made soluble bytruncation of and removal of all or a portion of the GPI anchor. In oneexample, the human hyaluronidase PH20, which is normally membraneanchored via a GPI anchor, can be made soluble by truncation of andremoval of all or a portion of the GPI anchor at the C-terminus.

Soluble hyaluronan degrading enzymes also include neutral active andacid active hyaluronidases. Depending on factors, such as, but notlimited to, the desired level of activity of the enzyme followingadministration and/or site of administration, neutral active and acidactive hyaluronidases can be selected. In a particular example, thehyaluronan degrading enzyme for use in the compositions, combinationsand methods herein is a soluble neutral active hyaluronidase.

Exemplary of a soluble hyaluronidase is PH20 from any species, such asany set forth in any of SEQ ID NOS: 1, 2, 11, 25, 27, 30, 31, 63-65 and185-186, or truncated forms thereof lacking all or a portion of theC-terminal GPI anchor, so long as the hyaluronidase is soluble andretains hyaluronidase activity. Also included among solublehyaluronidases are allelic variants or other variants of any of SEQ IDNOS:1, 2, 11, 25, 27, 30 31, 63-65 and 185-186, or truncated formsthereof. Allelic variants and other variants are known to one of skillin the art, and include polypeptides having 60%, 70%, 80%, 90%, 91%,92%, 93%, 94%, 95%. 96% 97% 98% or more sequence identity to any of SEQID NOS: 1, 2, 11, 25, 27, 30 31, 63-65 and 185-186, or truncated formsthereof. Amino acid variants include conservative and non-conservativemutations. It is understood that residues that are important orotherwise required for the activity of a hyaluronidase, such as anydescribed above or known to skill in the art, are generally invariant.These include, for example, active site residues. Thus, for example,amino acid residues 111, 113 and 176 (corresponding to residues in themature PH20 polypeptide set forth in SEQ ID NO:2) of a human PH20polypeptide, or soluble form thereof, are generally invariant and arenot altered. Other residues that confer glycosylation and formation ofdisulfide bonds required for proper folding also can be invariant.

In some instances, the soluble hyaluronan degrading enzyme is normallyGPI-anchored (such as, for example, human PH20) and is rendered solubleby truncation at the C-terminus. Such truncation can remove of all ofthe GPI anchor attachment signal sequence, or can remove only some ofthe GPI anchor attachment signal sequence. The resulting polypeptide,however, is soluble. In instances where the soluble hyaluronan degradingenzyme retains a portion of the GPI anchor attachment signal sequence,1, 2, 3, 4, 5, 6, 7 or more amino acid residues in the GPI-anchorattachment signal sequence can be retained, provided the polypeptide issoluble. Polypeptides containing one or more amino acids of the GPIanchor are termed extended soluble hyaluronan degrading enzymes. One ofskill in the art can determine whether a polypeptide is GPI-anchoredusing methods well known in the art. Such methods include, but are notlimited to, using known algorithms to predict the presence and locationof the GPI-anchor attachment signal sequence and ω-site, and performingsolubility analyses before and after digestion withphosphatidylinositol-specific phospholipase C (PI-PLC) or D (PI-PLD).

Extended soluble hyaluronan degrading enzymes can be produced by makingC-terminal truncations to any naturally GPI-anchored hyaluronandegrading enzyme such that the resulting polypeptide is soluble andcontains one or more amino acid residues from the GPI-anchor attachmentsignal sequence. Exemplary extended soluble hyaluronan degrading enzymesthat are C-terminally truncated but retain a portion of the GPI anchorattachment signal sequence include, but are not limited to, extendedsoluble PH20 (esPH20) polypeptides of primate origin, such as, forexample, human and chimpanzee esPH20 polypeptides. For example, theesPH20 polypeptides can be made by C-terminal truncation of any of themature or precursor polypeptides set forth in SEQ ID NOS:1, 2 or 185, orallelic or other variation thereof, including active fragment thereof,wherein the resulting polypeptide is soluble and retains one or moreamino acid residues from the GPI-anchor attachment signal sequence.Allelic variants and other variants are known to one of skill in theart, and include polypeptides having 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95% or more sequence identity to any of SEQ ID NOS: 1 or 2. TheesPH20 polypeptides provided herein can be C-terminally truncated by 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to the wild typepolypeptide, such as a polypeptide with a sequence set forth in SEQ IDNOS: 1, 2 or 185, provided the resulting esPH20 polypeptide is solubleand retains 1 or more amino acid residues from the GPI-anchor attachmentsignal sequence.

Typically, for use in the compositions, combinations and methods herein,a soluble human hyaluronan degrading enzyme, such as a soluble humanPH20, is used. Although hyaluronan degrading enzymes, such as PH20, fromother animals can be utilized, such preparations are potentiallyimmunogenic, since they are animal proteins. For example, a significantproportion of patients demonstrate prior sensitization secondary toingested foods, and since these are animal proteins, all patients have arisk of subsequent sensitization. Thus, non-human preparations may notbe suitable for chronic use. If non-human preparations are desired, itis contemplated herein that such polypeptides can be prepared to havereduced immunogenicity. Such modifications are within the level of oneof skill in the art and can include, for example, removal and/orreplacement of one or more antigenic epitopes on the molecule.

Hyaluronan degrading enzymes, including hyaluronidases (e.g., PH20),used in the methods herein can be recombinantly produced or can bepurified or partially-purified from natural sources, such as, forexample, from testes extracts. Methods for production of recombinantproteins, including recombinant hyaluronan degrading enzymes, areprovided elsewhere herein and are well known in the art.

a. Soluble Human PH20

Exemplary of a soluble hyaluronidase is soluble human PH20, Solubleforms of recombinant human PH20 have been produced and can be used inthe compositions, combinations and methods described herein. Theproduction of such soluble forms of PH20 is described in U.S. PublishedPatent Application Nos. US20040268425; US 20050260186 and US20060104968(incorporated by reference in their entirety), and in the Examples,below. For example, soluble PH20 polypeptides, include C-terminallytruncated variant polypeptides that include a sequence of amino acids inSEQ ID NO:1, or have at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%sequence identity to a sequence of amino acids included in SEQ ID NO:1,retain hyaluronidase activity and are soluble. Included among thesepolypeptides are soluble PH20 polypeptides that completely lack all or aportion of the GPI-anchor attachment signal sequence. Also included areextended soluble PH20 (esPH20) polypeptides that contain at least oneamino acid of the GPI anchor. Thus, instead of having a GPI-anchorcovalently attached to the C-terminus of the protein in the ER and beinganchored to the extracellular leaflet of the plasma membrane, thesepolypeptides are secreted and are soluble. C-terminally truncated PH20polypeptides can be C-terminally truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 5,60 or more amino acids compared to the full length wild typepolypeptide, such as a full length wild type polypeptide with a sequenceset forth in SEQ ID NOS:1 or 2, or allelic or species variants or othervariants thereof.

Exemplary C-terminally truncated human PH20 polypeptides provided hereininclude any having C-terminal truncations to generate polypeptidescontaining amino acid 1 to amino acid 465, 466, 467, 468, 469, 470, 471,472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, of thesequence of amino acids set forth in SEQ ID NO: 1, or correspondingpositions in an allelic or species variant thereof. When expressed inmammalian cells, the 35 amino acid N-terminal signal sequence is cleavedduring processing, and the mature form of the protein is secreted. Thus,exemplary mature C-terminally truncated soluble PH20 polypeptides cancontain amino acids 36 to 465, 466, 467, 468, 469, 470, 471, 472, 473,474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487,488, 489, 490, 491, 492, 493, 494, 495, 496, 497 of the sequence ofamino acids set forth in SEQ ID NO: 1 or corresponding positions in anallelic or species variant thereof. Table 4 provides non-limitingexamples of exemplary C-terminally truncated PH20 polypeptides,including C-terminally truncated soluble PH20 polypeptides. In Table 4below, the length (in amino acids) of the precursor and maturepolypeptides, and the sequence identifier (SEQ ID NO) in which exemplaryamino acid sequences of the precursor and mature polypeptides of theC-terminally truncated PH20 proteins are set forth, are provided. Thewild-type PH20 polypeptide also is included in Table 4 for comparison.

TABLE 4 Exemplary C-terminally truncated PH20 polypeptides Precursor(amino Precursor Mature Mature Polypeptide acids) SEQ ID NO (aminoacids) SEQ ID NO wildtype 509 1 474 2 SPAM1-FIVS 497 191 462 235SPAM1-MFIV 496 225 461 269 SPAM1-TMFI 495 192 460 236 SPAM1-ATMF 494 226459 270 SPAM1-SATM 493 193 458 237 SPAM1-LSAT 492 227 457 271 SPAM1-TLSA491 194 456 238 SPAM1-PSTL 489 195 454 239 SPAM1-SPST 488 228 453 272SPAM1-STLS 490 196 455 240 SPAM1-ASPS 487 197 452 241 SPAM1-NASP 486 229451 273 SPAM1-YNAS 485 198 450 242 SPAM1-FYNA 484 199 449 243 SPAM1-IFYN483 46 448 48 SPAM1-QIFY 482 3 447 4 SPAM1-PQIF 481 45 446 5 SPAM1-EPQI480 44 445 6 SPAM1-EEPQ 479 43 444 7 SPAM1-TEEP 478 42 443 8 SPAM1-ETEE477 41 442 9 SPAM1-METE 476 200 441 244 SPAM1-PMET 475 201 440 245SPAM1-PPME 474 202 439 246 SPAM1-KPPM 473 203 438 247 SPAM1-LKPP 472 204437 248 SPAM1-FLKP 471 205 436 249 SPAM1-AFLK 470 206 435 250 SPAM1-DAFL469 207 434 251 SPAM1-IDAF 468 208 433 252 SPAM1-CIDA 467 40 432 47SPAM1-VCID 466 209 431 253 SPAM1-GVCI 465 210 430 254

Soluble forms include, but are not limited to, any having C-terminaltruncations to generate polypeptides containing amino acids 1 to aminoacid 467, 477, 478, 479, 480, 481, 482 and 483 of the sequence of aminoacids set forth in SEQ ID NO:1. When expressed in mammalian cells, the35 amino acid N-terminal signal sequence is cleaved during processing,and the mature form of the protein is secreted. Thus, the mature solublepolypeptides contain amino acids 36 to 467, 477, 478, 479, 480, 481, 482and 483 of SEQ ID NO:1. Deletion mutants ending at amino acid position477 to 483 (corresponding to the precursor polypeptide set forth in SEQID NO:1) exhibit higher secreted hyaluronidase activity than the fulllength GPI-anchored form. Hence, exemplary of soluble hyaluronidasessoluble human PH20 polypeptides that are 442, 443, 444, 445, 446 or 447amino acids in length, such as set forth in any of SEQ ID NOS: 4-9, orallelic or species variants or other variants thereof.

Generally soluble forms of PH20 are produced using protein expressionsystems that facilitate correct N-glycosylation to ensure thepolypeptide retains activity, since glycosylation is important for thecatalytic activity and stability of hyaluronidases. Such cells include,for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).

b. rHuPH20

Recombinant soluble forms of human PH20 have been generated and can beused in the compositions, combinations and methods provided herein. Thegeneration of such soluble forms of recombinant human PH20 are describedin U.S. Published Patent Application Nos. US20040268425; US 20050260186and US20060104968, and in Examples 2-6, below. Exemplary of suchpolypeptides are those generated from a nucleic acid molecule encodingamino acids 1-482 (set forth in SEQ ID NO:3). Such an exemplary nucleicacid molecule is set forth in SEQ ID NO:49. Post translationalprocessing removes the 35 amino acid signal sequence, leaving a 447amino acid soluble recombinant human PH20 (SEQ ID NO:4). As produced inthe culture medium there is heterogeneity at the C-terminus such thatthe product, designated rHuPH20, includes a mixture of species that caninclude any one or more of SEQ ID NOS. 4-9 in various abundance.Typically, rHuPH20 is produced in cells that facilitate correctN-glycosylation to retain activity, such as CHO cells (e.g. DG44 CHOcells).

4. Glycosylation of Hyaluronan Degrading Enzymes

Glycosylation, including N- and O-linked glycosylation, of somehyaluronan degrading enzymes, including hyaluronidases, can be importantfor their catalytic activity and stability. While altering the type ofglycan modifying a glycoprotein can have dramatic affects on a protein'santigenicity, structural folding, solubility, and stability, mostenzymes are not thought to require glycosylation for optimal enzymeactivity. For some hyaluronidases, removal of N-linked glycosylation canresult in near complete inactivation of the hyaluronidase activity.Thus, for such hyaluronidases, the presence of N-linked glycans iscritical for generating an active enzyme.

N-linked oligosaccharides fall into several major types (oligomannose,complex, hybrid, sulfated), all of which have (Man)3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn residuesthat fall within-Asn-Xaa-Thr/Ser-sequences (where Xaa is not Pro).Glycosylation at an-Asn-Xaa-Cys-site has been reported for coagulationprotein C. In some instances, a hyaluronan degrading enzyme, such as ahyaluronidase, can contain both N-glycosidic and O-glycosidic linkages.For example, PH20 has O-linked oligosaccharides as well as N-linkedoligosaccharides. There are seven potential N-linked glycosylation sitesat N82, N166, N235, N254, N368, N393, N490 of human PH20 exemplified inSEQ ID NO: 1. As noted above, N-linked glycosylation at N490 is notrequired for hyaluronidase activity.

In some examples, the hyaluronan degrading enzymes for use in thecompositions, combinations and/or methods provided are glycosylated atone or all of the glycosylation sites. For example, for human PH20, or asoluble form thereof, 2, 3, 4, 5, or 6 of the N-glycosylation sitescorresponding to amino acids N82, N166, N235, N254, N368, and N393 ofSEQ ID NO: 1 are glycosylated. In some examples the hyaluronan degradingenzymes are glycosylated at one or more native glycosylation sites. Inother examples, the hyaluronan degrading enzymes are modified at one ormore non-native glycosylation sites to confer glycosylation of thepolypeptide at one or more additional site. In such examples, attachmentof additional sugar moieties can enhance the pharmacokinetic propertiesof the molecule, such as improved half-life and/or improved activity.

In other examples, the hyaluronan degrading enzymes for use in thecompositions, combinations and/or methods provided herein are partiallydeglycosylated (or N-partially glycosylated polypeptides). For example,partially deglycosylated soluble PH20 polypeptides that retain all or aportion of the hyaluronidase activity of a fully glycosylatedhyaluronidase can be used in the compositions, combinations and/ormethods provided herein. Exemplary partially deglycosylatedhyaluronidases include soluble forms of a partially deglycosylated PH20polypeptides from any species, such as any set forth in any of SEQ IDNOS: 1, 2, 11, 25, 27, 29, 30, 31, 32, 63, 65, 185 and 186, or allelicvariants, truncated variants, or other variants thereof. Such variantsare known to one of skill in the art, and include polypeptides having60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% or more sequence identity toany of SEQ ID NOS: 1, 2, 11, 25, 27, 29, 30, 31, 32, 63, 65, 185 and186, or truncated forms thereof. The partially deglycosylatedhyaluronidases provided herein also include hybrid, fusion and chimericpartially deglycosylated hyaluronidases, and partially deglycosylatedhyaluronidase conjugates.

Glycosidases, or glycoside hydrolases, are enzymes that catalyze thehydrolysis of the glycosidic linkage to generate two smaller sugars. Themajor types of N-glycans in vertebrates include high mannose glycans,hybrid glycans and complex glycans. There are several glycosidases thatresult in only partial protein deglycosylation, including: EndoF1, whichcleaves high mannose and hybrid type glycans; EndoF2, which cleavesbiantennary complex type glycans; EndoF3, which cleaves biantennary andmore branched complex glycans; and EndoH, which cleaves high mannose andhybrid type glycans. Treatment of a hyaluronan degrading enzyme, such asa soluble hyaluronidase, such as a soluble PH20, with one or all ofthese glycosidases can result in only partial deglycosylation and,therefore, retention of hyaluronidase activity.

Partially deglycosylated hyaluronan degrading enzymes, such as partiallydeglycosylated soluble hyaluronidases, can be produced by digestion withone or more glycosidases, generally a glycosidase that does not removeall N-glycans but only partially deglycosylates the protein. Forexample, treatment of PH20 (e.g. a recombinant PH20 designated rHuPH20)with one or all of the above glycosidases (e.g. EndoF1, EndoF2 and/orEndoF3) results in partial deglycosylation. These partiallydeglycosylated PH20 polypeptides can exhibit hyaluronidase enzymaticactivity that is comparable to the fully glycosylated polypeptides. Incontrast, treatment of PH20 with PNGaseF, a glycosidase that cleaves allN-glycans, results in complete removal of all N-glycans and therebyrenders PH20 enzymatically inactive. Thus, although all N-linkedglycosylation sites (such as, for example, those at amino acids N82,N166, N235, N254, N368, and N393 of human PH20, exemplified in SEQ IDNO: 1) can be glycosylated, treatment with one or more glycosidases canrender the extent of glycosylation reduced compared to a hyaluronidasethat is not digested with one or more glycosidases.

The partially deglycosylated hyaluronan degrading enzymes, includingpartially deglycosylated soluble PH20 polypeptides, can have 10%, 20%,30%, 40%, 50%, 60%, 70% or 80% of the level of glycosylation of a fullyglycosylated polypeptide. Typically, the partially deglycosylatedhyaluronan degrading enzymes, including partially deglycosylated solublePH20 polypeptides, exhibit hyaluronidase activity that is 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%,300%, 400%, 500%, 1000% or more of the hyaluronidase activity exhibitedby the fully glycosylated polypeptide.

5. Modifications of Hyaluronan Degrading Enzymes to Improve TheirPharmacokinetic Properties

Hyaluronan degrading enzymes can be modified to improve theirpharmacokinetic properties, such as increasing their half-life in vivoand/or activities. The modification of hyaluronan degrading enzymes foruse in the compositions, combinations and/or methods provided caninclude attaching, directly or indirectly via a linker, such ascovalently or by other stable linkage, a polymer, such as dextran, apolyethylene glycol (pegylation(PEG)) or sialyl moiety, or other suchpolymers, such as natural or sugar polymers.

Pegylation of therapeutics is known to increase resistance toproteolysis, increase plasma half-life, and decrease antigenicity andimmunogenicity. Covalent or other stable attachment (conjugation) ofpolymeric molecules, such as polyethylene glycol moiety (PEG), to thehyaluronan degrading enzyme thus can impart beneficial properties to theresulting enzyme-polymer composition. Such properties include improvedbiocompatibility, extension of protein (and enzymatic activity)half-life in the blood, cells and/or in other tissues within a subject,effective shielding of the protein from proteases and hydrolysis,improved biodistribution, enhanced pharmacokinetics and/orpharmacodynamics, and increased water solubility.

Exemplary polymers that can be conjugated to the hyaluronan degradingenzyme, include natural and synthetic homopolymers, such as polyols(i.e. poly-OH), polyamines (i.e. poly-NH2) and polycarboxyl acids (i.e.poly-COOH), and further heteropolymers i.e. polymers comprising one ormore different coupling groups e.g. a hydroxyl group and amine groups.Examples of suitable polymeric molecules include polymeric moleculesselected from among polyalkylene oxides (PAO), such as polyalkyleneglycols (PAG), including polyethylene glycols (PEG), methoxypolyethyleneglycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers(Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG) branched polyethyleneglycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates,polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acidanhydride, polystyrene-co-maleic acid anhydride, dextrans includingcarboxymethyl-dextrans, heparin, homologous albumin, celluloses,including methylcellulose, carboxymethylcellulose, ethylcellulose,hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose,hydrolysates of chitosan, starches such as hydroxyethyl-starches andhydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guargum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acidhydrolysates and bio-polymers.

Typically, the polymers are polyalkylene oxides (PAO), such aspolyethylene oxides, such as PEG, typically mPEG, which, in comparisonto polysaccharides such as dextran, pullulan and the like, have fewreactive groups capable of cross-linking. Typically, the polymers arenon-toxic polymeric molecules such as (m)polyethylene glycol (mPEG)which can be covalently conjugated to the hyaluronan degrading enzyme(e.g., to attachment groups on the protein surface) using a relativelysimple chemistry.

Suitable polymeric molecules for attachment to the hyaluronan degradingenzyme include, but are not limited to, polyethylene glycol (PEG) andPEG derivatives such as methoxy-polyethylene glycols (mPEG),PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG),branched PEGs, and polyethylene oxide (PEO) (see e.g. Roberts et al.,Advanced Drug Delivery Review 2002, 54: 459-476; Harris and Zalipsky, S(eds.) “Poly(ethylene glycol), Chemistry and Biological Applications”ACS Symposium Series 680, 1997; Mehvar et al., J. Pharm. Pharmaceut.Sci., 3(1):125-136, 2000; Harris, Nature Reviews 2:215 et seq. (2003);and Tsubery, J. Biol. Chem 279(37):38118-24, 2004). The polymericmolecule can be of a molecular weight typically ranging from about 3 kDato about 60 kDa. In some embodiments the polymeric molecule that isconjugated to a protein, such as rHuPH20, has a molecular weight of 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more than 60 kDa.

Various methods of modifying polypeptides by covalently attaching(conjugating) a PEG or PEG derivative (i.e. “PEGylation”) are known inthe art (see e.g., U.S. 2006/0104968; U.S. Pat. No. 5,672,662; U.S. Pat.No. 6,737,505; and U.S. 2004/0235734). Techniques for PEGylationinclude, but are not limited to, specialized linkers and couplingchemistries (see e.g., Roberts, Adv. Drug Deliv. Rev. 54:459-476, 2002),attachment of multiple PEG moieties to a single conjugation site (suchas via use of branched PEGs; see e.g., Guiotto et al., Bioorg. Med.Chem. Lett. 12:177-180, 2002), site-specific PEGylation and/ormono-PEGylation (see e.g., Chapman et al., Nature Biotech. 17:780-783,1999), and site-directed enzymatic PEGylation (see e.g., Sato, Adv. DrugDeliv. Rev., 54:487-504, 2002) (see, also, for example, Lu and Felix(1994) Int. J. Peptide Protein Res. 43:127-138; Lu and Felix (1993)Peptide Res. 6:140-6, 1993; Felix et al. (1995) Int. J. Peptide Res.46:253-64; Benhar et al. (1994) J. Biol. Chem. 269:13398-404; Brumeanuet al. (1995) J Immunol. 154:3088-95; see also, Caliceti et al. (2003)Adv. Drug Deliv. Rev. 55(10):1261-77 and Molineux (2003) Pharmacotherapy23 (8 Pt 2):3S-8S). Methods and techniques described in the art canproduce proteins having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10PEG or PEG derivatives attached to a single protein molecule (see e.g.,U.S. 2006/0104968).

Numerous reagents for PEGylation have been described in the art. Suchreagents include, but are not limited to, N-hydroxysuccinimidyl (NHS)activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEGsuccinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEGsuccinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acidsuccinimidyl ester, homobifunctional PEG-succinimidyl propionate,homobifunctional PEG propionaldehyde, homobifunctional PEGbutyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonatePEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEGbutryaldehyde, branched mPEG2 butyraldehyde, mPEG acetyl, mPEGpiperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinylsulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyldisulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylatePEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardiniet al., Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. BioactiveCompatible Polymers 12:197-207, 1997; U.S. Pat. No. 5,672,662; U.S. Pat.No. 5,932,462; U.S. Pat. No. 6,495,659; U.S. Pat. No. 6,737,505; U.S.Pat. No. 4,002,531; U.S. Pat. No. 4,179,337; U.S. Pat. No. 5,122,614;U.S. Pat. No. 5,183,550; U.S. Pat. No. 5,324,844; U.S. Pat. No.5,446,090; U.S. Pat. No. 5,612,460; U.S. Pat. No. 5,643,575; U.S. Pat.No. 5,766,581; U.S. Pat. No. 5,795,569; U.S. Pat. No. 5,808,096; U.S.Pat. No. 5,900,461; U.S. Pat. No. 5,919,455; U.S. Pat. No. 5,985,263;U.S. Pat. No. 5,990,237; U.S. Pat. No. 6,113,906; U.S. Pat. No.6,214,966; U.S. Pat. No. 6,258,351; U.S. Pat. No. 6,340,742; U.S. Pat.No. 6,413,507; U.S. Pat. No. 6,420,339; U.S. Pat. No. 6,437,025; U.S.Pat. No. 6,448,369; U.S. Pat. No. 6,461,802; U.S. Pat. No. 6,828,401;U.S. Pat. No. 6,858,736; U.S. 2001/0021763; U.S. 2001/0044526; U.S.2001/0046481; U.S. 2002/0052430; U.S. 2002/0072573; U.S. 2002/0156047;U.S. 2003/0114647; U.S. 2003/0143596; U.S. 2003/0158333; U.S.2003/0220447; U.S. 2004/0013637; US 2004/0235734; U.S. 2005/000360; U.S.2005/0114037; U.S. 2005/0171328; U.S. 2005/0209416; EP 1064951; EP0822199; WO 01076640; WO 0002017; WO 0249673; WO 9428024; and WO0187925).

In one example, the hyaluronan degrading enzyme for use in the methods,compositions, and combinations provided is a soluble hyaluronidase thatis PEGylated. In a particular example, the soluble hyaluronidase is aPEGylated PH20 hyaluronidase. In another particular example, the solublehyaluronidase is PEGylated rHuPH20, such as that described in Example10.

E. METHODS OF PRODUCING NUCLEIC ACIDS ENCODING AN INSULIN OR HYALURONANDEGRADING ENZYME AND POLYPEPTIDES THEREOF

Polypeptides of an insulin and hyaluronan degrading enzyme set forthherein, can be obtained by methods well known in the art for proteinpurification and recombinant protein expression. Polypeptides also canbe synthesized chemically. For example, the A-chain and B-chain ofinsulin can be chemically synthesized and then cross-linked by disulfidebonds through, for example, a reduction-reoxidation reaction. When thepolypeptides are produced by recombinant means, any method known tothose of skill in the art for identification of nucleic acids thatencode desired genes can be used. Any method available in the art can beused to obtain a full length (i.e., encompassing the entire codingregion) cDNA or genomic DNA clone encoding a hyaluronidase, such as froma cell or tissue source. Modified or variant insulins or hyaluronandegrading enzymes can be engineered from a wildtype polypeptide, such asby site-directed mutagenesis.

Polypeptides can be cloned or isolated using any available methods knownin the art for cloning and isolating nucleic acid molecules. Suchmethods include PCR amplification of nucleic acids and screening oflibraries, including nucleic acid hybridization screening,antibody-based screening and activity-based screening.

Methods for amplification of nucleic acids can be used to isolatenucleic acid molecules encoding a desired polypeptide, including forexample, polymerase chain reaction (PCR) methods. A nucleic acidcontaining material can be used as a starting material from which adesired polypeptide-encoding nucleic acid molecule can be isolated. Forexample, DNA and mRNA preparations, cell extracts, tissue extracts,fluid samples (e.g. blood, serum, saliva), samples from healthy and/ordiseased subjects can be used in amplification methods. Nucleic acidlibraries also can be used as a source of starting material. Primers canbe designed to amplify a desired polypeptide. For example, primers canbe designed based on expressed sequences from which a desiredpolypeptide is generated. Primers can be designed based onback-translation of a polypeptide amino acid sequence. Nucleic acidmolecules generated by amplification can be sequenced and confirmed toencode a desired polypeptide.

Additional nucleotide sequences can be joined to a polypeptide-encodingnucleic acid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a polypeptide-encoding nucleicacid molecule. Examples of such sequences include, but are not limitedto, promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences, for example heterologous signalsequences, designed to facilitate protein secretion. Such sequences areknown to those of skill in the art. Additional nucleotide residuessequences such as sequences of bases specifying protein binding regionsalso can be linked to enzyme-encoding nucleic acid molecules. Suchregions include, but are not limited to, sequences of residues thatfacilitate or encode proteins that facilitate uptake of an enzyme intospecific target cells, or otherwise alter pharmacokinetics of a productof a synthetic gene. For example, enzymes can be linked to PEG moieties.

In addition, tags or other moieties can be added, for example, to aid indetection or affinity purification of the polypeptide. For example,additional nucleotide residues sequences such as sequences of basesspecifying an epitope tag or other detectable marker also can be linkedto enzyme-encoding nucleic acid molecules. Exemplary of such sequencesinclude nucleic acid sequences encoding a His tag (e.g., 6×His, HHHHHH;SEQ ID NO:54) or Flag Tag (DYKDDDDK; SEQ ID NO:55).

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.). Other expression vectors include theHZ24 expression vector exemplified herein. The insertion into a cloningvector can, for example, be accomplished by ligating the DNA fragmentinto a cloning vector which has complementary cohesive termini.Insertion can be effected using TOPO cloning vectors (INVITROGEN,Carlsbad, Calif.). If the complementary restriction sites used tofragment the DNA are not present in the cloning vector, the ends of theDNA molecules can be enzymatically modified. Alternatively, any sitedesired can be produced by ligating nucleotide sequences (linkers) ontothe DNA termini; these ligated linkers can contain specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved vector andprotein gene can be modified by homopolymeric tailing. Recombinantmolecules can be introduced into host cells via, for example,transformation, transfection, infection, electroporation andsonoporation, so that many copies of the gene sequence are generated.

Insulin can be produced using a variety of techniques (see e.g. Ladischet al (1992) Biotechnol. Prog. 8:469-478). In some examples, nucleicacid encoding a preproinsulin or proinsulin polypeptide is inserted intoan expression vector. Upon expression, the preproinsulin or proinsulinpolypeptide is converted to insulin by enzymatic or chemical methodsthat cleave the signal sequence and/or the C peptide, resulting in theA- and B-chains that are cross-linked by disulfide bonds through, forexample, a reduction-reoxidation reaction (see e.g. Cousens et al.,(1987) Gene 61:265-275, Chance et al., (1993) Diabetes Care 4:147-154).In another example, the nucleic acid encoding the A-chain and B-chain ofan insulin are inserted into one or two expression vectors forco-expression as a single polypeptide from one expression vector orexpression as two polypeptides from one or two expression vectors. Thus,the A- and B-chain polypeptides can be expressed separately and thencombined to generate an insulin, or can be co-expressed, in the absenceof a C chain. In instances where the A- and B-chains are co-expressed asa single polypeptide, the nucleic acid encoding the subunits also canencode a linker or spacer between the B-chain and A-chain, such as alinker or spacer described below. The nucleic acid inserted into theexpression vector can contain, for example, nucleic acid encoding theinsulin B-chain, a linker, such as for example, analanine-alanine-lysine linker, and the A-chain, resulting in expressionof, for example, “insulin B chain-Ala-Ala-Lys-insulin A chain.”

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

1. Vectors and Cells

For recombinant expression of one or more of the desired proteins, suchas any described herein, the nucleic acid containing all or a portion ofthe nucleotide sequence encoding the protein can be inserted into anappropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedprotein coding sequence. The necessary transcriptional and translationalsignals also can be supplied by the native promoter for enzyme genes,and/or their flanking regions.

Also provided are vectors that contain a nucleic acid encoding theenzyme. Cells containing the vectors also are provided. The cellsinclude eukaryotic and prokaryotic cells, and the vectors are anysuitable for use therein.

Prokaryotic and eukaryotic cells, including endothelial cells,containing the vectors are provided. Such cells include bacterial cells,yeast cells, fungal cells, Archea, plant cells, insect cells and animalcells. The cells are used to produce a protein thereof by growing theabove-described cells under conditions whereby the encoded protein isexpressed by the cell, and recovering the expressed protein. Forpurposes herein, for example, the enzyme can be secreted into themedium.

Provided are vectors that contain a sequence of nucleotides that encodesthe soluble hyaluronidase polypeptide coupled to the native orheterologous signal sequence, as well as multiple copies thereof. Thevectors can be selected for expression of the enzyme protein in the cellor such that the enzyme protein is expressed as a secreted protein.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding protein, or domains,derivatives, fragments or homologs thereof, can be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins can be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for a desired protein. Promoterswhich can be used include but are not limited to the SV40 early promoter(Bernoist and Chambon, Nature 290:304-310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), theregulatory sequences of the metallothionein gene (Brinster et al.,Nature 296:39-42 (1982)); prokaryotic expression vectors such as theβ-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA80:21-25 (1983)); see also “Useful Proteins from Recombinant Bacteria”:in Scientific American 242:79-94 (1980)); plant expression vectorscontaining the nopaline synthetase promoter (Herrara-Estrella et al.,Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNApromoter (Gardner et al., Nucleic Acids Res. 9:2871 (1981)), and thepromoter of the photosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7:425-515 (1987)); insulin gene control region which isactive in pancreatic beta cells (Hanahan et al., Nature 315:115-122(1985)), immunoglobulin gene control region which is active in lymphoidcells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444(1987)), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell45:485-495 (1986)), albumin gene control region which is active in liver(Pinkert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., Mol.Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)),alpha-1 antitrypsin gene control region which is active in liver (Kelseyet al., Genes and Devel. 1:161-171 (1987)), beta globin gene controlregion which is active in myeloid cells (Magram et al., Nature315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basicprotein gene control region which is active in oligodendrocyte cells ofthe brain (Readhead et al., Cell 48:703-712 (1987)), myosin lightchain-2 gene control region which is active in skeletal muscle (Shani,Nature 314:283-286 (1985)), and gonadotrophic releasing hormone genecontrol region which is active in gonadotrophs of the hypothalamus(Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a desired protein, or adomain, fragment, derivative or homolog, thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pQEexpression vectors (available from Qiagen, Valencia, Calif.; see alsoliterature published by Qiagen describing the system). pQE vectors havea phage T5 promoter (recognized by E. coli RNA polymerase) and a doublelac operator repression module to provide tightly regulated, high-levelexpression of recombinant proteins in E. coli, a synthetic ribosomalbinding site (RBS II) for efficient translation, a 6×His tag codingsequence, t₀ and T1 transcriptional terminators, ColE1 origin ofreplication, and a beta-lactamase gene for conferring ampicillinresistance. The pQE vectors enable placement of a 6×His tag at eitherthe N- or C-terminus of the recombinant protein. Such plasmids includepQE 32, pQE 30, and pQE 31 which provide multiple cloning sites for allthree reading frames and provide for the expression of N-terminally6×His-tagged proteins. Other exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pETexpression vectors (see, U.S. Pat. No. 4,952,496; available fromNOVAGEN, Madison, Wis.; see, also literature published by Novagendescribing the system). Such plasmids include pET 11a, which containsthe T7lac promoter, T7 terminator, the inducible E. coli lac operator,and the lac repressor gene; pET 12a-c, which contains the T7 promoter,T7 terminator, and the E. coli ompT secretion signal; and pET 15b andpET19b (NOVAGEN, Madison, Wis.), which contain a His-Tag™ leadersequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn, the T7-lac promoter region and the T7 terminator.

Exemplary of a vector for mammalian cell expression is the HZ24expression vector. The HZ24 expression vector was derived from the pCIvector backbone (Promega). It contains DNA encoding the Beta-lactamaseresistance gene (AmpR), an F1 origin of replication, a Cytomegalovirusimmediate-early enhancer/promoter region (CMV), and an SV40 latepolyadenylation signal (SV40). The expression vector also has aninternal ribosome entry site (IRES) from the ECMV virus (Clontech) andthe mouse dihydrofolate reductase (DHFR) gene.

2. Linker Moieties

In some examples, insulin is prepared by generating the A-chain andB-chain polypeptides with a linker, such that, for example, theC-terminus of the B-chain is joined to the N-terminus of the A-chain bya short linker. The A-chain and B-chains can be expressed from a singlepolypeptide containing a linker, or can be expressed separately and thenjoined by a linker. The linker moiety is selected depending upon theproperties desired. The linker moiety should be long enough and flexibleenough to allow the A-chain and B-chain to mimic the naturalconformation of the insulin. Linkers can be any moiety suitable to theinsulin A-chain and B-chain. Such moieties include, but are not limitedto, peptidic linkages; amino acid and peptide linkages, typicallycontaining between one and about 60 amino acids; chemical linkers, suchas heterobifunctional cleavable cross-linkers, photocleavable linkersand acid cleavable linkers.

The linker moieties can be peptides. The peptide typically has fromabout 2 to about 60 amino acid residues, for example from about 5 toabout 40, or from about 10 to about 30 amino acid residues. Peptidiclinkers can conveniently be encoded by nucleic acid and incorporated infusion proteins upon expression in a host cell, such as E. coli. In oneexample, an alanine-alanine-lysine (AAK) (SEQ ID NO:178) linker isencoded in a nucleic acid between nucleic acid encoding the insulinB-chain and nucleic acid encoding the A-chain, such that uponexpression, an “insulin B-chain-AAK-insulin A chain” polypeptide isproduced. Peptide linkers can be a flexible spacer amino acid sequence,such as those known in single-chain antibody research. Examples of suchknown linker moieties include, but are not limited to, RPPPPC (SEQ IDNO:166) or SSPPPPC (SEQ ID NO:167), GGGGS (SEQ ID NO:168), (GGGGS)_(n)(SEQ. ID NO:169), GKSSGSGSESKS (SEQ ID NO:170), GSTSGSGKSSEGKG (SEQ. IDNO:171), GSTSGSGKSSEGSGSTKG (SEQ ID NO:172), GSTSGSGKSSEGKG (SEQ IDNO:173), GSTSGSGKPGSGEGSTKG (SEQ ID NO:174), EGKSSGSGSESKEF (SEQ IDNO:175), SRSSG (SEQ. ID NO:176) and SGSSC (SEQ ID NO:177).

Alternatively, the peptide linker moiety can be VM (SEQ ID NO: 179) orAM (SEQ ID NO: 180), or have the structure described by the formula:AM(G_(2 to 4)S)_(x)AM wherein X is an integer from 1 to 11 (SEQ ID NO:181). Additional linking moieties are described, for example, in Hustonet al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883; Whitlow, M. etal., (1993) Protein Engineering 6:989-995; Newton et al. (1996)Biochemistry 35:545-553; A. J. Cumber et al. (1992) Bioconj. Chem.3:397-401; Ladurner et al. (1997) J. Mol. Biol. 273:330-337; and U.S.Pat. No. 4,894,443.

In some examples, peptide linkers are encoded by nucleic acid andincorporated between the B-chain and A-chain upon expression in a hostcell, such as E. coli or S. cerevisiae. In other examples, a peptidelinker is synthesized by chemical methods. This can be performed in aseparate protocol to the synthesis of one or more of the A- and B-chain,after which the components are joined, such as with the use ofheterobifunctional linkers. Alternatively, a peptide linker can besynthesized at the N- or C-terminus of one of the insulin chains, whichis then linked to the other chain via the peptide linker, such as with aheterobifunctional linker.

Any linker known to those of skill in the art can be used herein to linkthe insulin A-chain and B-chain. Linkers and linkages that are suitablefor chemically linking the chains include, but are not limited to,disulfide bonds, thioether bonds, hindered disulfide bonds, and covalentbonds between free reactive groups, such as amine and thiol groups.These bonds are produced using heterobifunctional reagents to producereactive thiol groups on one or both of the polypeptides and thenreacting the thiol groups on one polypeptide with reactive thiol groupsor amine groups to which reactive maleimido groups or thiol groups canbe attached on the other. Other linkers include, acid cleavable linkers,such as bismaleimideothoxy propane, acid labile-transferrin conjugatesand adipic acid diihydrazide, that would be cleaved in more acidicintracellular compartments; cross linkers that are cleaved upon exposureto UV or visible light and linkers, such as the various domains, such asCH1, CH2, and CH3, from the constant region of human IgG1 (see, Batra etal. (1993) Molecular Immunol. 30:379-386). In some embodiments, severallinkers can be included in order to take advantage of desired propertiesof each linker. Chemical linkers and peptide linkers can be inserted bycovalently coupling the linker to the insulin A-chain and B-chain. Theheterobifunctional agents, described below, can be used to effect suchcovalent coupling. Peptide linkers also can be linked by expressing DNAencoding the linker between the B-chain and A-chain.

Other linkers that can be used to join the A-chain and B-chain ofinsulin include: enzyme substrates, such as cathepsin B substrate,cathepsin D substrate, trypsin substrate, thrombin substrate, subtilisinsubstrate, Factor Xa substrate, and enterokinase substrate; linkers thatincrease solubility, flexibility, and/or intracellular cleavabilityinclude linkers, such as (gly_(m)ser)_(n) and (ser_(m)gly)_(n), in whichm is 1 to 6, preferably 1 to 4, more preferably 2 to 4, and n is 1 to30, preferably 1 to 10, more preferably 1 to 4 (see, e.g., InternationalPCT application No. WO 96/06641, which provides exemplary linkers). Insome embodiments, several linkers can be included in order to takeadvantage of desired properties of each linker.

3. Expression

Insulin and hyaluronan degrading enzyme polypeptides can be produced byany method known to those of skill in the art including in vivo and invitro methods. Desired proteins can be expressed in any organismsuitable to produce the required amounts and forms of the proteins, suchas for example, needed for administration and treatment. Expressionhosts include prokaryotic and eukaryotic organisms such as E. coli,yeast, plants, insect cells, mammalian cells, including human cell linesand transgenic animals. Expression hosts can differ in their proteinproduction levels as well as the types of post-translationalmodifications that are present on the expressed proteins. The choice ofexpression host can be made based on these and other factors, such asregulatory and safety considerations, production costs and the need andmethods for purification.

Many expression vectors are available and known to those of skill in theart and can be used for expression of proteins. The choice of expressionvector will be influenced by the choice of host expression system. Ingeneral, expression vectors can include transcriptional promoters andoptionally enhancers, translational signals, and transcriptional andtranslational termination signals. Expression vectors that are used forstable transformation typically have a selectable marker which allowsselection and maintenance of the transformed cells. In some cases, anorigin of replication can be used to amplify the copy number of thevector.

Soluble hyaluronidase polypeptides also can be utilized or expressed asprotein fusions. For example, an enzyme fusion can be generated to addadditional functionality to an enzyme. Examples of enzyme fusionproteins include, but are not limited to, fusions of a signal sequence,a tag such as for localization, e.g. a his₆ tag or a myc tag, or a tagfor purification, for example, a GST fusion, and a sequence fordirecting protein secretion and/or membrane association.

a. Prokaryotic Cells

Prokaryotes, especially E. coli, provide a system for producing largeamounts of proteins. Transformation of E. coli is a simple and rapidtechnique well known to those of skill in the art. Expression vectorsfor E. coli can contain inducible promoters, such promoters are usefulfor inducing high levels of protein expression and for expressingproteins that exhibit some toxicity to the host cells. Examples ofinducible promoters include the lac promoter, the trp promoter, thehybrid tac promoter, the T7 and SP6 RNA promoters and the temperatureregulated λPL promoter.

Proteins, such as any provided herein, can be expressed in thecytoplasmic environment of E. coli. The cytoplasm is a reducingenvironment and for some molecules, this can result in the formation ofinsoluble inclusion bodies. Reducing agents such as dithiothreotol andβ-mercaptoethanol and denaturants, such as guanidine-HCl and urea can beused to resolubilize the proteins. An alternative approach is theexpression of proteins in the periplasmic space of bacteria whichprovides an oxidizing environment and chaperonin-like and disulfideisomerases and can lead to the production of soluble protein. Typically,a leader sequence is fused to the protein to be expressed which directsthe protein to the periplasm. The leader is then removed by signalpeptidases inside the periplasm. Examples of periplasmic-targetingleader sequences include the pelB leader from the pectate lyase gene andthe leader derived from the alkaline phosphatase gene. In some cases,periplasmic expression allows leakage of the expressed protein into theculture medium. The secretion of proteins allows quick and simplepurification from the culture supernatant. Proteins that are notsecreted can be obtained from the periplasm by osmotic lysis. Similar tocytoplasmic expression, in some cases proteins can become insoluble anddenaturants and reducing agents can be used to facilitate solubilizationand refolding. Temperature of induction and growth also can influenceexpression levels and solubility, typically temperatures between 25° C.and 37° C. are used. Typically, bacteria produce aglycosylated proteins.Thus, if proteins require glycosylation for function, glycosylation canbe added in vitro after purification from host cells.

b. Yeast Cells

Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are wellknown yeast expression hosts that can be used for production ofproteins, such as any described herein. Yeast can be transformed withepisomal replicating vectors or by stable chromosomal integration byhomologous recombination. Typically, inducible promoters are used toregulate gene expression. Examples of such promoters include GAL1, GAL7and GAL5 and metallothionein promoters, such as CUP1, AOX1 or otherPichia or other yeast promoter. Expression vectors often include aselectable marker such as LEU2, TRP1, HIS3 and URA3 for selection andmaintenance of the transformed DNA. Proteins expressed in yeast areoften soluble. Co-expression with chaperonins such as Bip and proteindisulfide isomerase can improve expression levels and solubility.Additionally, proteins expressed in yeast can be directed for secretionusing secretion signal peptide fusions such as the yeast mating typealpha-factor secretion signal from Saccharomyces cerevisae and fusionswith yeast cell surface proteins such as the Aga2p mating adhesionreceptor or the Arxula adeninivorans glucoamylase. A protease cleavagesite such as for the Kex-2 protease, can be engineered to remove thefused sequences from the expressed polypeptides as they exit thesecretion pathway. Yeast also is capable of glycosylation atAsn-X-Ser/Thr motifs.

c. Insect Cells

Insect cells, particularly using baculovirus expression, are useful forexpressing polypeptides such as hyaluronidase polypeptides. Insect cellsexpress high levels of protein and are capable of most of thepost-translational modifications used by higher eukaryotes. Baculovirushave a restrictive host range which improves the safety and reducesregulatory concerns of eukaryotic expression. Typical expression vectorsuse a promoter for high level expression such as the polyhedrin promoterof baculovirus. Commonly used baculovirus systems include thebaculoviruses such as Autographa californica nuclear polyhedrosis virus(AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and aninsect cell line such as Sf9 derived from Spodoptera frugiperda,Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1). For high-levelexpression, the nucleotide sequence of the molecule to be expressed isfused immediately downstream of the polyhedrin initiation codon of thevirus. Mammalian secretion signals are accurately processed in insectcells and can be used to secrete the expressed protein into the culturemedium. In addition, the cell lines Pseudaletia unipuncta (A7S) andDanaus plexippus (DpN1) produce proteins with glycosylation patternssimilar to mammalian cell systems.

An alternative expression system in insect cells is the use of stablytransformed cells. Cell lines such as the Schneider 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

d. Mammalian Cells

Mammalian expression systems can be used to express proteins includingsoluble hyaluronidase polypeptides. Expression constructs can betransferred to mammalian cells by viral infection such as adenovirus orby direct DNA transfer such as liposomes, calcium phosphate,DEAE-dextran and by physical means such as electroporation andmicroinjection. Expression vectors for mammalian cells typically includean mRNA cap site, a TATA box, a translational initiation sequence (Kozakconsensus sequence) and polyadenylation elements. IRES elements also canbe added to permit bicistronic expression with another gene, such as aselectable marker. Such vectors often include transcriptionalpromoter-enhancers for high-level expression, for example the SV40promoter-enhancer, the human cytomegalovirus (CMV) promoter and the longterminal repeat of Rous sarcoma virus (RSV). These promoter-enhancersare active in many cell types. Tissue and cell-type promoters andenhancer regions also can be used for expression. Exemplarypromoter/enhancer regions include, but are not limited to, those fromgenes such as elastase I, insulin, immunoglobulin, mouse mammary tumorvirus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta globin,myelin basic protein, myosin light chain 2, and gonadotropic releasinghormone gene control. Selectable markers can be used to select for andmaintain cells with the expression construct. Examples of selectablemarker genes include, but are not limited to, hygromycin Bphosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyltransferase, aminoglycoside phosphotransferase, dihydrofolate reductase(DHFR) and thymidine kinase. For example, expression can be performed inthe presence of methotrexate to select for only those cells expressingthe DHFR gene. Fusion with cell surface signaling molecules such asTCR-ζ and Fc_(ε)RI-γ can direct expression of the proteins in an activestate on the cell surface. Many cell lines are available for mammalianexpression including mouse, rat human, monkey, chicken and hamstercells. Exemplary cell lines include but are not limited to CHO,Balb/3T3, HeLa, MT2, mouse NS0 (nonsecreting) and other myeloma celllines, hybridoma and heterohybridoma cell lines, lymphocytes,fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Celllines also are available adapted to serum-free media which facilitatespurification of secreted proteins from the cell culture media. Examplesinclude CHO-S cells (Invitrogen, Carlsbad, Calif., cat #11619-012) andthe serum free EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng.84:332-42.). Cell lines also are available that are adapted to grow inspecial media optimized for maximal expression. For example, DG44 CHOcells are adapted to grow in suspension culture in a chemically defined,animal product-free medium.

e. Plants

Transgenic plant cells and plants can be used to express proteins suchas any described herein. Expression constructs are typically transferredto plants using direct DNA transfer such as microprojectile bombardmentand PEG-mediated transfer into protoplasts, and withagrobacterium-mediated transformation. Expression vectors can includepromoter and enhancer sequences, transcriptional termination elementsand translational control elements. Expression vectors andtransformation techniques are usually divided between dicot hosts, suchas Arabidopsis and tobacco, and monocot hosts, such as corn and rice.Examples of plant promoters used for expression include the cauliflowermosaic virus promoter, the nopaline synthase promoter, the ribosebisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters.Selectable markers such as hygromycin, phosphomannose isomerase andneomycin phosphotransferase are often used to facilitate selection andmaintenance of transformed cells. Transformed plant cells can bemaintained in culture as cells, aggregates (callus tissue) orregenerated into whole plants. Transgenic plant cells also can includealgae engineered to produce hyaluronidase polypeptides. Because plantshave different glycosylation patterns than mammalian cells, this caninfluence the choice of protein produced in these hosts.

4. Purification Techniques

Method for purification of polypeptides, including insulin andhyaluronan degrading enzyme polypeptides or other proteins, from hostcells will depend on the chosen host cells and expression systems. Forsecreted molecules, proteins are generally purified from the culturemedia after removing the cells. For intracellular expression, cells canbe lysed and the proteins purified from the extract. When transgenicorganisms such as transgenic plants and animals are used for expression,tissues or organs can be used as starting material to make a lysed cellextract. Additionally, transgenic animal production can include theproduction of polypeptides in milk or eggs, which can be collected, andif necessary, the proteins can be extracted and further purified usingstandard methods in the art.

Proteins, such as insulin polypeptides or hyaluronan degrading enzymepolypeptides, can be purified using standard protein purificationtechniques known in the art including but not limited to, SDS-PAGE, sizefractionation and size exclusion chromatography, ammonium sulfateprecipitation and ionic exchange chromatography, such as anion exchangechromatography. Affinity purification techniques also can be utilized toimprove the efficiency and purity of the preparations. For example,antibodies, receptors and other molecules that bind hyaluronidaseenzymes can be used in affinity purification. Expression constructs alsocan be engineered to add an affinity tag to a protein such as a mycepitope, GST fusion or His₆ and affinity purified with myc antibody,glutathione resin and Ni-resin, respectively. Purity can be assessed byany method known in the art including gel electrophoresis, orthoganalHPLC methods, staining and spectrophotometric techniques.

F. PREPARATION, FORMULATION AND ADMINISTRATION OF INSULIN AND HYALURONANDEGRADING ENZYME POLYPEPTIDES

Pharmaceutical compositions of fast-acting insulin and hyaluronandegrading enzymes are provided herein for administration. Hyaluronandegrading enzymes are co-formulated or co-administered withpharmaceutical formulations of fast-acting insulin to enhance thedelivery of fast-acting insulin to the blood by increasing the rate ofabsorption and increasing the bioavailability of insulin. Increased rateof absorption and bioavailability can be achieved, for example, byreversible depolymerization of hyaluronan by the hyaluronan degradingenzyme, which temporarily (typically for a period of less than 24 hours)increases the hydraulic conductivity of the subcutaneous space. Thus,hyaluronan degrading enzymes can be used to achieve elevated and/or morerapidly achieved concentrations of the insulin following parenteral,such as, for example, subcutaneous, administration compared toconventional methods of subcutaneous administration, to provide, forexample, a more potent and/or more rapid response for a given dose.Co-administration of a hyaluronan degrading enzyme with a fast-actinginsulin, therefore, can render the fast-acting insulin a superfast-acting insulin. The hyaluronan degrading enzymes also can be usedto achieve glycemic control with a lower dose of administered insulin.The ability of hyaluronan degrading enzymes to enhance bulk fluid flowat and near a site of injection or infusion also can improve otheraspects of associated pharmacologic delivery. For example, the increasein bulk fluid flow can help to allow the volume of fluid injected to bemore readily dispersed from the site of injection (reducing potentiallypainful or other adverse consequences of injection). This isparticularly important for subcutaneous infusions to permit higher dosesto be administered.

Thus, by virtue of the increased rate of absorption,parenterally-administered fast-acting insulins, can become superfast-acting insulins when administered with a hyaluronan degradingenzyme. The advantages over administration of insulin without ahyaluronan degrading enzyme is that co-administered or co-formulatedhyaluronan degrading enzyme/insulin can result in more favorable dosingregimens, for example, lower insulin doses and/or the use of moreeffective closed loop systems, and improved therapeutic effects, forexample, more efficient glycemic control and/or reduced excess insulin.For example, by lowering the dose, side effects associated with excesscirculating insulin, such as observed with higher doses of insulin, canbe reduced. Such side effects include, but are not limited to,hypoglycemia and obesity.

The compositions can be formulated in lyophilized or liquid form. Wherethe compositions are provided in lyophilized form they can bereconstituted just prior to use by an appropriate solution, for example,a sterile saline solution or sterile water for injection. Thecompositions can be provided together or separately. For example, thefast-acting insulin and hyaluronan degrading enzyme can be co-formulatedin a single composition, or can be provided as separate compositions.When provided as separate compositions, the hyaluronan degrading enzymeand insulin can be packaged for administration together, sequentially orintermittently. The combinations can be packaged as a kit.

1. Formulations

The compounds can be formulated into any suitable pharmaceuticalpreparations for parenteral administration such as solutions,suspensions, sustained release formulations, or powders. Typically, thecompounds are formulated into pharmaceutical compositions usingtechniques and procedures well known in the art (see e.g., AnselIntroduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126).Pharmaceutically acceptable compositions are prepared in view ofapprovals from a regulatory agency or other agency prepared inaccordance with generally recognized pharmacopeia for use in animals andin humans. The formulation should suit the mode of administration.

Pharmaceutical compositions can include carriers such as a diluent,adjuvant, excipient, or vehicle with which a hyaluronan degrading enzymeand insulin is administered. Examples of suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin. Such compositions will contain a therapeutically effectiveamount of the compound, generally in purified form or partially purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the patient. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, and sesame oil. Water is a typical carrierwhen the pharmaceutical composition is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions also can beemployed as liquid carriers, particularly for injectable solutions.Compositions can contain along with an active ingredient: a bulkingagent such as lactose, sucrose, dicalcium phosphate, orcarboxymethylcellulose; a lubricant, such as magnesium stearate, calciumstearate and talc; and a binder such as starch, natural gums, such asgum acaciagelatin, glucose, molasses, polyvinylpyrrolidine, cellulosesand derivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, glycerin,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, and ethanol. A composition, if desired, also can contain minoramounts of wetting or emulsifying agents, or pH buffering agents, forexample, acetate, sodium citrate, glycerin, cyclodextrine derivatives,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and other such agents.

Pharmaceutical therapeutically active compounds and derivatives thereofare typically formulated and administered in unit dosage forms ormultiple dosage forms. Each unit dose contains a predetermined quantityof therapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit dose forms can be administered in fractions or multiples thereof. Amultiple dose form is a plurality of identical unit dosage formspackaged in a single container to be administered in segregated unitdose form. Examples of multiple dose forms include vials, cartridges,bottles of tablets or capsules or bottles of pints or gallons. Hence,multiple dose form is a multiple of unit doses that are not segregatedin packaging. Generally, dosage forms or compositions containing activeingredient in the range of 0.005% to 100% with the balance made up froma non-toxic carrier can be prepared.

Compositions provided herein typically are formulated for administrationby subcutaneous route, although other routes of administration arecontemplated, such as any route known to those of skill in the artincluding intramuscular, intraperitoneal, intravenous, intradermal,intralesional, intraperitoneal injection, epidural, vaginal, rectal,local, otic, transdermal administration or any route. Formulationssuited for such routes are known to one of skill in the art.Administration can be local, topical or systemic depending upon thelocus of treatment. Local administration to an area in need of treatmentcan be achieved by, for example, but not limited to, local infusionduring surgery, topical application, e.g., in conjunction with a wounddressing after surgery, by injection, by means of a catheter, by meansof a suppository, or by means of an implant. Compositions also can beadministered with other biologically active agents, either sequentially,intermittently or in the same composition.

The most suitable route in any given case depends on a variety offactors, such as the nature of the disease, the tolerance of the subjectto a particular administration route, the severity of the disease, andthe particular composition that is used. Typically, the compositionsprovided herein are administered parenterally. In some examples,hyaluronan degrading enzymes are administered so that they reach theinterstitium of skin or tissues, thereby degrading the interstitialspace for subsequent delivery of insulin. Thus, in some examples, directadministration under the skin, such as by subcutaneous administrationmethods, is contemplated. Thus, in one example, local administration canbe achieved by injection, such as from a syringe or insulin pen or otherarticle of manufacture containing an injection device such as a needle.In another example, local administration can be achieved by infusion,which can be facilitated by the use of a pump or other similar device.Other modes of administration also are contemplated. Pharmaceuticalcompositions can be formulated in dosage forms appropriate for eachroute of administration.

Subcutaneous administration, generally characterized by injection orinfusion, is contemplated herein. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. The pharmaceutical compositions cancontain other minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents, stabilizers,solubility enhancers, and other such agents, such as for example, sodiumacetate, sodium phosphate, sorbitan monolaurate, triethanolamine oleateand cyclodextrins. In some examples, zinc, calcium, serum albumin, EDTA,calcium chloride and/or phenolic preservatives are included in thecompositions. The percentage of active compound contained in suchcompositions is highly dependent on the specific nature thereof, as wellas the activity of the compound and the needs of the subject.

Injectables are designed for local and systemic administration. Forpurposes herein, local administration is desired for directadministration to the affected interstitium. Preparations for parenteraladministration include sterile solutions ready for injection, steriledry soluble products, such as lyophilized powders, ready to be combinedwith a solvent just prior to use, including hypodermic tablets, sterilesuspensions ready for injection, sterile dry insoluble products ready tobe combined with a vehicle just prior to use and sterile emulsions. Thesolutions can be either aqueous or nonaqueous. If administeredintravenously, suitable carriers include physiological saline orphosphate buffered saline (PBS), and solutions containing thickening andsolubilizing agents, such as glucose, polyethylene glycol, andpolypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers, which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN 80). A sequestering or chelating agent of metalions include EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles andsodium hydroxide, hydrochloric acid, citric acid or lactic acid for pHadjustment.

The concentration of the pharmaceutically active compound is adjusted sothat an injection or infusion provides an effective amount to producethe desired pharmacological effect, such as glycemic control. The exactdose depends on the age, weight and condition of the patient or animalas is known in the art. The unit-dose parenteral preparations can bepackaged in, for example, an ampoule, a cartridge, a vial or a syringewith a needle. The volume of liquid solution or reconstituted powderpreparation, containing the pharmaceutically active compound, is afunction of the disease to be treated and the particular article ofmanufacture chosen for package. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

In one example, pharmaceutical preparation can be in liquid form, forexample, solutions, syrups or suspensions. If provided in liquid form,the pharmaceutical preparations can be provided as a concentratedpreparation to be diluted to a therapeutically effective concentrationbefore use. Such liquid preparations can be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). In another example, pharmaceutical preparations can bepresented in lyophilized form for reconstitution with water or othersuitable vehicle before use.

The fast-acting insulin and hyaluronan degrading enzyme compositions canbe co-formulated as a single composition, or can be provided as twoseparate compositions. When provided as two compositions, thecompositions can be mixed prior to administration to be co-administered,or can be kept separated and then co administered together, sequentiallyor intermittently. In some examples, the fast-acting insulin andhyaluronan degrading enzyme are co-formulated as super fast-actinginsulin compositions. As discussed below, the compositions can beformulated for single or multiple dosage, wherein the dosages can beprovided as a ratio of amount of a hyaluronan degrading enzyme toinsulin administered. For example, a hyaluronan degrading enzyme can beadministered at 1 hyaluronidase U/insulin U (1:1) to 50:1 or more, forexample, at or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,11:1, 12:1, 13:1, 14:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1or more. In other examples, lower ratios of hyaluronan degrading enzymeto insulin are administered, including, for example, 1 hyaluronidase U/2insulin U (1:2), 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15 or 1:20.The fast-acting insulin can be present in the co-formulated or separatecompositions in concentrations of or about 10 U/mL, 20 U/mL, 30 U/mL, 40U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, 100 U/mL, 150 U/mL,200 U/mL, 250 U/mL, 300 U/mL, 350 U/mL, 400 U/mL, 450 U/ml or 500 U/mL.Typically, the amount of hyaluronan degrading enzyme in theco-formulated or separate compositions is functionally equivalent to orto at least 1 U/mL, 2 U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8U/mL, 9 U/mL, 10 U/mL, 15 U/mL, 20 U/mL or 25 U/mL of hyaluronidaseactivity. In some examples, the amount of hyaluronan degrading enzyme inthe co-formulated or separate compositions is functionally equivalent toor to at least 30 or 35 U/ml, of hyaluronidase activity, such as or 30U/mL, 35 U/mL, 37.5 U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL,90 U/mL, 100 U/mL, 200 U/mL, 300 U/mL, 400 U/mL, 500 U/mL, 600 U/mL, 700U/mL, 800 U/mL, 900 U/mL, 1000 U/ml, 2000 U/mL, 3000 U/mL or 5000 U/mLof hyaluronidase activity.

The super fast-acting insulin compositions provided herein can containone or more pH buffers (such as, for example, histidine, phosphate, Trisor other buffers), or acidic buffer (such as acetate, citrate, pyruvate,Gly-HCl, succinate, lactate, maleate or other buffers), tonicitymodifier (such as, for example, an amino acid, polyalcohol, glycerol,NaCl, trehalose, other salts and/or sugars), stabilizer (such as sodiumbenzoate to stabilize insulin), chelating agent, such asethylenediaminetetraacetic acid, ethylenediaminetetraacetate or calciumEDTA, oxygen scavenger, such as methionine, ascorbic acid/ascorbate,citric acid/citrate, or albumin, and/or a preservative, such aspreservative containing an aromatic ring (e.g. phenol or cresol).Exemplary preservatives that are useful in the compositions providedherein include, but are not limited to, m-cresol, phenol and paraben orany combination thereof. In some examples, m-cresol is added at orapproximately 0.05% to 0.2%, such as 0.1% to 0.15% (e.g. at or about0.1%, 0.11%, 0.12%, 0.13%, 0.14% or 0.15%). Suitable concentrations ofphenol or paraben include 0.05-0.25%, such as 0.1% to 0.2% (e.g. at orabout 0.1%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% and0.2%). Typically, NaCl or other salt is provided in compositionscontaining a hyaluronan degrading enzyme. Exemplary concentrations ofNaCl include 50 mM to 200 mM, such as 50 mM to 150 mM, including 50 mM,60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM and 150 mMNaCl. In some examples, to retain the stability of the compositionsprovided herein, as the salt concentration in the composition isincreased, so too is the pH. Glycerol also can be included as a tonicitymodifier and/or to increase the viscosity of the compositions.

Exemplary stabilizers that are useful for compositions containing ahyaluronan degrading enzyme include detergents or surfactants, such aspolysorbates and proteins such as human serum albumin. In some examples,one or more surfactants (e.g. such as Pluronic F68) are included in thecompositions, such as at or about 0.001% to 0.1%, typically at or about0.005% to 0.03% (e.g. 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%,0.012%, 0.014%, 0.016%, 0.018%, 0.02% or 0.03. Exemplary concentrationsof serum albumin that are useful in the compositions herein include 0.1mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL,0.8 mg/mL, 0.9 mg/mL or 1 mg/mL, but can be more or less. Polysorbatesalso can be present in the compositions at, for example, concentrationsof or about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%,0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%,0.09% or 0.1%. Exemplary stabilizers that are useful for compositionscontaining an insulin include zinc and m-cresol. For example, zinc canfunction to stabilize the insulin hexamer. Zinc can be provided, forexample, as zinc oxide, zinc acetate or zinc chloride. Zinc can bepresent in a composition provided herein at between or about 0.001 to0.1 mg per 100 units of insulin, such as 0.002 milligrams per 100 unitsof insulin (mg/100 U), 0.005 mg/100 U, 0.01 mg/100 U, 0.012 mg/100 U,0.014 mg/100 U, 0.016 mg/100 U, 0.017 mg/100 U, 0.018 mg/100 U, 0.02mg/100 U, 0.022 mg/100 U, 0.024 mg/100 U, 0.026 mg/100 U, 0.28 mg/100 U,0.03 mg/100 U, 0.04 mg/100 U, 0.05 mg/100 U, 0.06 mg/100 U, 0.07 mg/100U, 0.08 mg/100 U or 0.1 mg/100 U. In one example, zinc is present at0.017 mg per 100 U insulin. A metal chelating agent, such as calciumEDTA (CaEDTA), also can be present, such as for example, atconcentrations of between approximately 0.02 mM to 20 mM, such as 0.02mM, 0.04 mM, 0.06 mM, 0.08 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM,0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM or more.In some examples, when both a chelating agent and zinc are present in acomposition provided herein, the chelating agent is present inapproximately equal amounts (i.e. 0.6 to 1.4 molar ratio) or molarexcess to zinc, such as for example, at a ratio of or about 2:1, 5:1,10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1 or morechelating agent:zinc. Calcium chloride also can be included in thecompositions at, for example, between about 0.2 mM to 20 mM.

In some instances, any one or more of the components described above arepresent in only the fast-acting insulin composition or the hyaluronandegrading composition, until the two compositions are eitherco-formulated or delivered to the subject as a super fast-acting insulincomposition. For example, the fast-acting insulin composition cancontain zinc at between or about 0.001 to 0.1 mg per 100 units ofinsulin, such as 0.002 milligrams per 100 units of insulin (mg/100 U),0.005 mg/100 U, 0.01 mg/100 U, 0.012 mg/100 U, 0.014 mg/100 U, 0.016mg/100 U, 0.017 mg/100 U, 0.018 mg/100 U, 0.02 mg/100 U, 0.022 mg/100 U,0.024 mg/100 U, 0.026 mg/100 U, 0.28 mg/100 U, 0.03 mg/100 U, 0.04mg/100 U, 0.05 mg/100 U, 0.06 mg/100 U, 0.07 mg/100 U, 0.08 mg/100 U or0.1 mg/100 U and no chelating agent, such as EDTA, while the hyaluronandegrading composition can contain a chelating agent, such as EDTA, at orabout 0.02 mM to 20 mM, such as 0.02 mM, 0.04 mM, 0.06 mM, 0.08 mM, 0.1mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1mM, 5 mM, 10 mM, 15 mM, 20 mM or more and no zinc. Thus, it is only whenthe two compositions are mixed, such as when being co-formulated orco-administered, that the composition containing insulin also containsEDTA, and the composition containing a hyaluronan degrading enzyme alsocontains zinc. In some instances, the initial fast-acting insulincomposition and hyaluronan degrading enzyme composition containsufficient amounts of zinc or chelating agent, respectively, that whenmixed for co-formulation or co-administration, the chelating agent ispresent in approximately equimolar amounts (i.e. 0.6 to 1.4 molar ratio)or molar excess to zinc, such as for example, at a ratio of or about2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1,100:1 or more chelating agent:zinc.

The pH and the osmolarity of the compositions can be adjusted by one ofskill in the art to optimize the conditions for the desired activity andstability of the composition. For example, as noted above, in someinstances, if the salt concentration is increased, the pH also can beincreased to retain stability of the composition. Further, one of skillin the art can change the pH to increase solubility of the particularfast-acting insulin used in the super fast-acting insulins providedherein. In some examples, the compositions provided herein that containone or both of a fast-acting insulin and a hyaluronan degrading enzymehave an osmolarity of at or about 100 mOsm/kg, 120 mOsm/kg, 140 mOsm/kg,160 mOsm/kg, 180 mOsm/kg, 200 mOsm/kg, 220 mOsm/kg, 240 mOsm/kg, 260mOsm/kg, 280 mOsm/kg, 300 mOsm/kg, 320 mOsm/kg, 340 mOsm/kg, 360mOsm/kg, 380 mOsm/kg, 400 mOsm/kg, 420 mOsm/kg, 440 mOsm/kg, 460mOsm/kg, 500 or more mOsm/kg, and a pH of at or about 6, 6.2, 6.4, 6.6,6.8, 7, 7.2, 7.4, 7.6, 7.8 or 8. In some examples, the pH from or fromabout 6.5 to or to about 7.5, such as 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4 or 7.5.

Administration methods can be employed to decrease the exposure ofselected compounds to degradative processes, such as proteolyticdegradation and immunological intervention via antigenic and immunogenicresponses. Examples of such methods include local administration at thesite of treatment. Pegylation of therapeutics has been reported toincrease resistance to proteolysis, increase plasma half-life, anddecrease antigenicity and immunogenicity. Examples of pegylationmethodologies are known in the art (see for example, Lu and Felix, Int.J. Peptide Protein Res., 43: 127-138, 1994; Lu and Felix, Peptide Res.,6: 140-146, 1993; Felix et al., Int. J. Peptide Res., 46: 253-64, 1995;Benhar et al., J. Biol. Chem., 269: 13398-404, 1994; Brumeanu et al., JImmunol., 154: 3088-95, 1995; see also, Caliceti et al. (2003) Adv. DrugDeliv. Rev. 55(10):1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt2):3S-8S). Pegylation also can be used in the delivery of nucleic acidmolecules in vivo. For example, pegylation of adenovirus can increasestability and gene transfer (see, e.g., Cheng et al. (2003) Pharm. Res.20(9): 1444-51).

Lyophilized Powders

Of interest herein are lyophilized powders, which can be reconstitutedfor administration as solutions, emulsions and other mixtures. They alsocan be reconstituted and formulated as solids or gels.

The sterile, lyophilized powder is prepared by dissolving an activecompound in a buffer solution. The buffer solution can contain anexcipient which improves the stability or other pharmacologicalcomponent of the powder or reconstituted solution, prepared from thepowder. Subsequent sterile filtration of the solution followed bylyophilization under standard conditions known to those of skill in theart provides the desired formulation. Briefly, the lyophilized powder isprepared by dissolving an excipient, such as dextrose, sorbital,fructose, corn syrup, xylitol, glycerin, glucose, sucrose or othersuitable agent, in a suitable buffer, such as citrate, Tris, histidine,sodium or potassium phosphate or other such buffer known to those ofskill in the art. Then, a selected enzyme is added to the resultingmixture, and stirred until it dissolves. The resulting mixture issterile filtered or treated to remove particulates and to insuresterility, and apportioned into vials for lyophilization. Each vial willcontain a single dosage or multiple dosages of the compound. Thelyophilized powder can be stored under appropriate conditions, such asat about 4° C. to room temperature. Reconstitution of this lyophilizedpowder with a buffer solution provides a formulation for use inparenteral administration.

2. Dosage and Administration

The hyaluronan degrading enzyme provided herein can be formulated aspharmaceutical compositions for single dosage or multiple dosageadministration. For example, the compositions provided herein cancontain hyaluronan degrading enzyme at 1 hyaluronidase U/insulin U (1:1)to 50:1 or more, for example, at or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 20:1, 25:1, 30:1,35:1, 40:1, 45:1, 50:1 or more. In other examples, lower ratios ofhyaluronan degrading enzyme to insulin are provided in the compositions,including, for example, 1 hyaluronidase U/2 insulin U (1:2), 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15 or 1:20. The selected hyaluronandegrading enzyme is included in an amount sufficient to exert atherapeutically useful effect in the absence of undesirable side effectson the patient treated. The therapeutically effective concentration canbe determined empirically by testing the polypeptides in known in vitroand in vivo systems such as by using the assays provided herein or knownin the art (see e.g., Taliani et al. (1996) Anal. Biochem., 240: 60-67;Filocamo et al. (1997) J Virology, 71: 1417-1427; Sudo et al. (1996)Antiviral Res. 32: 9-18; Buffard et al. (1995) Virology, 209:52-59;Bianchi et al. (1996) Anal. Biochem., 237: 239-244; Hamatake et al.(1996) Intervirology 39:249-258; Steinkuhler et al. (1998) Biochem.,37:8899-8905; D'Souza et al. (1995) J Gen. Virol., 76:1729-1736;Takeshita et al. (1997) Anal. Biochem., 247:242-246; see also e.g,Shimizu et al. (1994) J. Virol. 68:8406-8408; Mizutani et al. (1996) J.Virol. 70:7219-7223; Mizutani et al. (1996) Biochem. Biophys. Res.Commun., 227:822-826; Lu et al. (1996) Proc. Natl. Acad. Sci. (USA),93:1412-1417; Hahm et al., (1996) Virology, 226:318-326; Ito et al.(1996) J. Gen. Virol., 77:1043-1054; Mizutani et al. (1995) Biochem.Biophys. Res. Commun., 212:906-911; Cho et al. (1997) J. Virol. Meth.65:201-207) and then extrapolated therefrom for dosages for humans.

Therapeutically effective dosages for the super fast-acting insulincompositions containing a fast-acting insulin and a hyaluronan degradingenzyme can be determined based upon, for example, pharmacokinetic (PK)data and pharmacodynamic (PD) data, such as described below and inExample 1, and the known therapeutic doses of the fast-acting insulinwhen delivered without a hyaluronan degrading enzyme. Changes in insulinconcentration in blood or plasma or serum with time provides informationon in vivo (1) absorption for parenteral administration; (2)distribution, and (3) elimination of insulin. A pharmacokinetic modeldefines these physiological changes in the concentration of insulin as afunction of time (that is,

$\left. \frac{\left( {X} \right)}{\left( {t} \right)} \right)$

and characterizes them mathematically using rates and volumes. The modelparameters thus derived for insulin will remain relatively constantuntil a perturbation occurs. A well established PK model for insulin canprovide reasonable predictions of exposure (which is closely related toefficacy and for drug safety that results from exaggerate pharmacology).Clinical decisions on dose selection and dose schedule of insulin can befacilitated and justified using PK modeling and simulations. Apharmacodynamic model serves a similar purpose for prediction ofclinical outcome.

Typically, a therapeutically effective dose of a hyaluronan degradingenzyme is at or about 0.3 Units (U) to 5,000 U of a hyaluronan degradingenzyme. For example, a hyaluronan degrading enzyme can be administeredsubcutaneously at or about 0.3 U, 0.5 U, 1 U, 2 U, 3 U, 5 U, 10 U, 20 U,30 U, 40 U, 50 U, 100 U, 150 U, 200 U, 250 U, 300 U, 350 U, 400 U, 450U, 500 U, 600 U, 700 U, 800 U, 900 U, 1000 U, 2,000 U, 3,000 U, 4,000Units, 5,000 U or more. In some examples, dosages can be provided as aratio of amount of a hyaluronan degrading enzyme to insulinadministered. For example, a hyaluronan degrading enzyme can beadministered at 1 hyaluronidase U/insulin U (1:1) to 50:1 or more, forexample, at or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,11:1, 12:1, 13:1, 14:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1or more. In other examples, lower ratios of hyaluronan degrading enzymeto insulin are administered, including, for example, 1 hyaluronidase U/2insulin U (1:2), 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15 or 1:20.Typically, volumes of injections or infusions of hyaluronan degradingenzyme contemplated herein are from at or about 0.01 mL, 0.05 mL, 0.1mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL,7 mL, 8 mL, 9 ml, 10 ml or more. The hyaluronan degrading enzyme can beprovided as a stock solution at or about 1 U/mL, 2 U/mL, 3 U/mL, 4 U/mL,5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL, 15 U/mL, 20 U/mL, 25U/mL, 30 U/mL, 35 U/mL, 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90U/mL 100 U/ml, 150 U/ml, 200 U/ml, 300 U/ml, 400 U/ml, 500 U/mL, 600U/mL, 800 U/mL or 1000 U/mL, or can be provided in a more concentratedform, for example at or about 2000 U/ml, 3000 Units/ml, 4000 U/ml, 5000U/ml, 8000 U/ml, 10,000 U/mL or 20,000 U/mL for use directly or fordilution to the effective concentration prior to use. The hyaluronandegrading enzyme can be provided as a liquid or lyophilized formulation.

The insulin preparations provided herein can be formulated aspharmaceutical compositions for single or multiple dose use. Forexample, in some instances, insulin preparations are formulated forsingle dose administration in an amount sufficient to providepost-prandial glycemic control. In other examples, insulin preparationsare formulated for multiple dose administration or multi use vials, suchas for use in an insulin pen, insulin pump or other continuous insulindelivery device, or closed loop system. The insulin preparations can beprovided in lyophilized or liquid form as discussed elsewhere herein.

The insulin can be provided in a therapeutically effective dose.Therapeutically effective doses can be determined empirically by testingthe compounds in known in vitro and in vivo systems, such as the assaysprovided herein, and also can be individualized for each subject basedupon such factors as metabolism, food intake and severity of thedisease. The concentration of a selected insulin in the compositiondepends on, for example, absorption, inactivation and excretion rates ofthe complex, the physicochemical characteristics of the complex, thedosage schedule, and amount administered as well as other factors knownto those of skill in the art. For example, it is understood that theprecise dosage of treatment is a function of the blood glucose levels ina subject, and can be determined empirically using known algorithms orby extrapolation from in vivo or in vitro test data, past experience ofthe subject, carbohydrate counting to determine the carbohydrate contentin a meal and, therefore, the estimated prandial blood glucose increaseand subsequent requirement for insulin. It is to be noted thatconcentrations and dosage values can vary with each subject treated. Itis to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the formulations, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope thereof. The amount of a selected insulinpreparation to be administered for the treatment of a diabetic conditioncan be determined by standard clinical techniques. In addition, in vitroassays and animal models can be employed to help identify optimal dosageranges.

Hence, the precise dosage, which can be determined empirically, candepend on the particular insulin preparation, the regime and dosingschedule with hyaluronan degrading enzyme, the route of administration,the type of diabetes to be treated, the seriousness of the disease andthe subject being treated. Generally, insulin is provided in an amountthat achieves glycemic control. For example, to achieve post prandialglycemic control, diabetic subjects typically are administered a bolusinjection of or about 0.05 U of fast-acting insulin per kg body weight(U/kg) to 1.0 U/kg 30 minutes to 5 minutes prior to a meal, when insulinis delivered without a hyaluronan degrading enzyme. It is understoodthat this dose can be increased or decreased as appropriate based upon,for example, the metabolism of a particular subject, the content of themeal, and blood glucose levels. It is further understood that the timeat which the insulin is delivered for post prandial glycemic control canbe changed to be closer to or further from the time of ingestion of ameal, and, in some cases, can be changed such that the insulin isdelivered at the time of the meal or after the meal. A subject can,therefore, be administered a super fast-acting insulin compositionprovided herein by administering an insulin, such as a fast-actinginsulin, in combination with a hyaluronan degrading enzyme, at a doselower than that administered when insulin is administered alone and/orat a time closer to ingestion of a meal compared to the time at whichthe insulin alone dose is typically administered.

Fast-acting insulins typically are administered at doses of between 0.05Units/kg to 0.25 Units/kg, such as, for example, 0.10 Units/kg, althoughthe particular dose varies. Super fast-acting insulin compositions canbe administered at lower doses compared to the fast-acting insulinadministered in the absence of a hyaluronan degrading enzyme. Asdiscussed elsewhere herein, the degree to which the amount of afast-acting insulin can be lowered by administering it as a super fastacting insulin composition varies, depending on, for example, the typeof diabetes the patient has. Typically, the reduction in the amount offast-acting insulin administered to Type 2 diabetic patients whenadministered as a super fast-acting insulin composition is greater thanthe reduction in the amount of fast-acting insulin administered to Type1 diabetic patients when administered as a super fast-acting insulincomposition. For example, in instances where a Type 1 diabetic patientand Type 2 diabetic patient are both administered 0.20 U/kg offast-acting insulin to control postprandial glucose levels, the Type 1diabetic patient can be administered 0.15 U/kg of fast-acting insulin ina super fast-acting insulin composition to achieve the same or betterglycemic control, and the Type 2 diabetic patient can be administered0.10 U/kg fast-acting insulin in a super fast-acting insulin compositionto achieve the same or better glycemic control. Thus, in some examples,it is contemplated herein that the amount of a fast-acting insulin thatis administered with a hyaluronan degrading enzyme as a superfast-acting insulin to a Type 2 diabetic patient to achieve glycemiccontrol can be reduced by, for example, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80% or more compared to the amount required forglycemic control when administered without a hyaluronan degradingenzyme, and the amount of a fast-acting insulin that is administeredwith a hyaluronan degrading enzyme as a super fast-acting insulincomposition to a Type 1 diabetic patient to achieve glycemic control canbe reduced by, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70% or more compared to the amount required for glycemiccontrol when administered without a hyaluronan degrading enzyme.

Exemplary dosage ranges for parenteral, such as subcutaneous,administration of insulin using the methods and compositions providedherein to control postprandial blood glucose levels are from at or about0.05 U/kg to 0.50 U/kg, such as 0.05 U/kg, 0.06 U/kg, 0.07 U/kg, 0.08U/kg, 0.09 U/kg, 0.10 U/kg, 0.11 U/kg, 0.12 U/kg, 0.13 U/kg, 0.14 U/kg,0.15 U/kg, 0.20 U/kg, 0.25 U/kg, 0.30 U/kg, 0.40 U/kg, 0.50 U/kg or 1.0U/kg. The particular dosage and formulation thereof depends upon thedisease and individual. If necessary dosage can be empiricallydetermined. To achieve such dosages, volumes of insulin preparationsadministered subcutaneously to control postprandial glucose levels canbe at or about 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 75 μL, 100 μL, 150 μL,200 μL, 250 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL,1000 μL or more. For example, a 100 U/mL insulin formulation forindications described herein can be subcutaneously administered to a 70kg subject in a volume of 35 μL to 350 μL to achieve a dosage of 0.05U/kg to 0.50 U/kg of insulin. The compositions and methods providedherein also can be administered to diabetic subjects to effect glycemiccontrol throughout the day and night, in addition to postprandialglycemic control. Typically, dosages of insulin administered to providecontinuous glycemic control are less than those required to achievepostprandial glycemic control. Dosages can, however, be increased ordecreased based on blood glucose levels. Exemplary dosage ranges forparenteral, such as subcutaneous, administration of insulin using themethods and compositions provided herein to provide continuous glycemiccontrol are from at or about 0.001 U/kg to 0.30 U/kg, such as 0.001U/kg, 0.005 U/kg, 0.01 U/kg, 0.02 U/kg, 0.05 U/kg to 0.30 U/kg, such as0.05 U/kg, 0.06 U/kg, 0.07 U/kg, 0.08 U/kg, 0.09 U/kg, 0.10 U/kg, 0.11U/kg, 0.12 U/kg, 0.13 U/kg, 0.14 U/kg, 0.15 U/kg, 0.20 U/kg, 0.25 U/kg,0.30 U/kg, 0.40 U/kg, 0.50 U/kg or 1.0 U/kg. The particular dosage andformulation thereof depends upon the disease, the time ofadministration, and the individual. If necessary dosage can beempirically determined. The dosage for an individual is typicallytitrated down to the minimal dosage required to achieve a therapeuticeffect, such as the minimal dosage required to achieve glycemic control.The amount of insulin sufficient to achieve glycemic control can bedetermined empirically, such as by glucose challenge.

The hyaluronan degrading enzyme can be administered prior, subsequently,intermittently or simultaneously to the insulin preparation. Generally,the hyaluronan degrading enzyme is administered prior to orsimultaneously with administration of the insulin preparation to permitthe hyaluronan degrading enzyme to degrade the hyaluronic acid in theinterstitial space. In one example, the insulin composition andhyaluronan degrading enzyme composition are co-formulated and,therefore, administered simultaneously. In another example, thehyaluronan degrading enzyme composition is administered prior to theinsulin composition, such as 1 minute, 2 minutes, 3 minutes, 4 minutes,5 minutes or more prior to administration of the insulin preparation. Insome examples, the hyaluronidase is administered together with theinsulin preparation. As will be appreciated by those of skill in theart, the desired proximity of co-administration can be readily optimizedby testing the effects of administering the agents at varying times insuitable models, such as in suitable animal models.

Both the insulin preparation and the hyaluronan degrading enzymepreparation can be administered at once, or can be divided into a numberof smaller doses to be administered at intervals of time. Selectedinsulin preparations can be administered in one or more doses over thecourse of a treatment time for example over several minutes, hours,days, weeks, or months. In some cases, continuous administration isuseful. It is understood that the precise dosage and course ofadministration depends on the indication and patient's tolerability.

Also, it is understood that the precise dosage and duration of treatmentis a function of the diabetes being treated and can be determinedempirically using known testing protocols or by extrapolation from invivo or in vitro test data. It is to be noted that concentrations anddosage values also can vary with the severity of the diabetes and otherfactors, such as metabolism, food intake, and body weight of thesubject. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or use of compositions andcombinations containing them. The compositions can be administered everyminute, every several minutes, hourly, daily, weekly, monthly, yearly oronce, depending upon the subject and the diabetic state. Generally,dosage regimens are chosen to limit toxicity and/or other negativeeffects, such as excess insulin. It should be noted that the attendingphysician would know how to and when to terminate, interrupt or adjusttherapy to lower dosage. Conversely, the attending physician would alsoknow how to and when to adjust treatment to higher levels if theclinical response is not adequate (precluding toxic side effects).

Mode of Administration

a. Syringes

The compositions provided herein can be parentally administered to asubject using one or more of several modes of administration, including,but not limited to, syringes, insulin pens, insulin pumps, or in thecontext of a closed loop system or any combination thereof. For example,single-use syringes, including insulin syringes, can be used toadminister discrete bolus injections of the compositions. Thecompositions can be administered using the same syringe, such as whenthe insulin and hyaluronan degrading enzyme preparations areco-formulated, or can be administered sequentially using differentsyringes. Syringes useful for administrations of the compositionsprovided herein include insulin syringes, which can be designed to holdstandard concentrations of insulin preparations, including 100 U/mlconcentrations of insulin preparations, and have markings in insulinunits for ease of administration. In other examples, any one or more ofan insulin syringe or insulin pump or similar device is used toadminister one or both of the insulin preparation and the hyaluronandegrading enzyme preparation.

b. Insulin Pen

An insulin pen is a delivery system that can be used to administer thecompositions provided herein. Insulin pens include those withreplaceable cartridges filled with the composition to be administeredand those with non-replaceable cartridges. Insulin pens withnon-replaceable cartridges are typically disposed of when the cartridgehas been emptied. Insulin pens enable dosing in, for example, half unit,one unit or two unit increments, which are generally measured using adosing dial or other mechanism to set the dose (see e.g. U.S. Pat. Nos.5,947,934, 6,074,372, 6,110,149, 6,524,280, 6,582,404). The compositionis then delivered by way of a fine needle attached to the pen. Insulinpens are well known in the art and include those described elsewhere,including, but not limited to, those described in U.S. Pat. Nos.5,947,934, 4,973,318, 5,462,535, 5,599,323, 5,626,566, 5,984,906,6,074,372, 6,110,149, 6,302,869, 6,379,339 and 7,241,278). Other similardosing devices, such as for example, those described in U.S. Pat. Nos.5,947,394, 6,074,372, 6,110,149 and 6,379,339, also can be used toadminister the compositions provided herein, either as a co-formulationof insulin and hyaluronan degrading enzyme or separately as an insulincomposition and a hyaluronan degrading enzyme composition. In someexamples, the insulin pen or similar device also contains a sensor ormonitor than can measure the blood glucose level of the subject (seee.g. WO2003047426).

Insulin pens and similar delivery devices that can be used, or modifiedto be used, to deliver the insulin compositions provided herein are wellknown in the art and include, but are not limited to, those marketedunder the trademarks Autopen® (Owen Mumford, Inc.), Disetronic Pen(Disetronic Medical Systems), Humalog® Pen (Eli Lilly and Company),Humalog® Mix 75/25 Pen (Eli Lilly and Company), Humulin® 70/30 Pen (EliLilly and Company), Humulin® N Pen (Eli Lilly and Company), Novolog®FlexPen (Novo Nordisk), NovoPen® 3 (Novo Nordisk), NovoPen® 4 (NovoNordisk), NovoPen® Junior (Novo Nordisk), Novolog® Mix 70/30 FlexPen(Novo Nordisk), InDuo® (Novo Nordisk), Novolin® InnoLet® (Novo Nordisk),Innovo® (Novo Nordisk), OptiPen® (Sanofi-Aventis) OptiPen® Pro2(Sanofi-Aventis), OptiSet® (Sanofi-Aventis) and SoloSTAR®(Sanofi-Aventis).

c. Insulin Pumps and Other Insulin Delivery Devices

The compositions provided herein can be administered to a diabeticsubject using an insulin delivery device, such as an insulin pump orother similar continuous infusion device. Insulin delivery devicestypically contain at least one disposable reservoir containing aninsulin composition, a pump (including any controls, software,processing modules and/or batteries) and a disposable infusion set,including a cannula or needle for subcutaneous injection and a tubeconnecting the cannula or needle to the insulin reservoir. For use witha super fast-acting insulin composition, the insulin delivery device cancontain one reservoir containing a co-formulated insulin and hyaluronandegrading enzyme compositions, or can contain one or more reservoirs,such that the fast-acting insulin and hyaluronan degrading enzymecompositions are contained in the same or separate reservoirs. In suchinstances, the insulin delivery device can deliver each compositionsimultaneously or subsequent to each other. Thus, such devices can beused to administer the super fast-acting insulin compositions providedherein. The compositions can be administered continuously or in bolusinjections. Further, an insulin delivery device user has the ability toinfluence the profile of the insulin by shaping the bolus. For example,a standard bolus can be administered, which is an infusion similar to adiscrete injection in that all of the dose is pumped immediately. Anextended bolus is a slow infusion over time that avoids a high initialdose and extends the action of the composition. A combination boluscontaining both a standard bolus and an extended bolus also can beadministered using an insulin pump or other continuous delivery system.Insulin delivery devices are known in the art and described elsewhere,including, but not limited to, in U.S. Pat. Nos. 6,554,798, 6,641,533,6,744,350, 6,852,104, 6,872,200, 6,936,029, 6,979,326, 6,999,854,7,025,713 and 7,109,878. Insulin delivery devices also can be connectedto a glucose monitor or sensor, and/or can contain a means to calculatethe recommended insulin dose based upon blood glucose levels,carbohydrate content of a meal, or other input. Further insulin deliverydevices can be implantable or can be external to the subject.

d. Closed Loop Systems

Closed loop systems, sometimes referred to as an artificial pancreas,are of particular interest for use with the compositions and methodsprovided herein. Closed loop systems refer to systems with an integratedcontinuous glucose monitor, an insulin pump or other delivery system andcontroller that includes a mathematical algorithm that constantlycalculates the required insulin infusion for glycemic control based uponreal time measurements of blood glucose levels. Such systems, whenoptimized, can facilitate constant and very tight glycemic control,similar to the natural insulin response and glycemic control observed ina healthy non-diabetic subject. To be effective, however, closed loopsystems require both a reliable and accurate continuous glucose monitor,and delivery of an insulin with a very fast action. For example, delaysin insulin absorption and action associated with subcutaneous deliveryof fast-acting insulins can lead to large postprandial glycemicexcursions (Hovorka et al. (2006) Diabetic Med. 23:1-12). The delaybecause of insulin absorption, insulin action, interstitial glucosekinetics, and the transport time for ex vivo-based monitoring systems,such as those based on the microdialysis technique, can result in anoverall 100 minute or more time lag from the time of insulin delivery tothe peak of its detectable glucose-lowering effect (Hovorka et al.(2006) Diabetic Med. 23:1-12). Thus, once administered, insulin willcontinue to increase its measurable effect for nearly 2 hours. This cancomplicate effective lowering of glucose concentration following mealingestion using a closed-loop system. First, a glucose increase has tobe detected. However, this typically happens only after an approximate10-40 minute lag. The system must determine that a meal has beendigested and administer an appropriate insulin dose. The ability of thesystem to compensate subsequently for a ‘misjudged’ insulin dose iscompromised by long delays and the inability to ‘withdraw’ insulin onceadministered. Such problems can, at least in part, be overcome by usinga super fast-acting insulin composition, such as those provided herein,which exhibit an increased rate and level of absorption and anassociated improvement in the pharmacodynamics (as described in Example1, below). The super fast-acting insulin compositions provided hereinhave a reduced t_(max) (i.e. achieve maximal concentration faster) thanfast-acting insulins and begin controlling blood glucose levels fasterthan fast-acting insulins. This increased rate of absorbance and onsetof action reduces the lag between insulin action and glucose monitoringand input, resulting in a more effective closed loop system that canmore tightly control blood glucose levels, reducing glycemic excursions.

Closed loop systems are well known in the art and have been describedelsewhere, including, but not limited to, U.S. Pat. Nos. 5,279,543,5,569,186, 6,558,351, 6,558,345, 6,589,229, 6,669,663, 6,740,072,7,267,665 and 7,354,420, which are incorporated by reference herein.These and similar systems, easily identifiable by one of skill in theart, can be used to deliver the super fast-acting insulin compositionsprovided herein. Closed loops systems include a sensor system to measureblood glucose levels, a controller and a delivery system. Thisintegrated system is designed to model a pancreatic beta cell (β-cell),such that it controls an infusion device to deliver insulin into asubject in a similar concentration profile as would be created by fullyfunctioning human β-cells when responding to changes in blood glucoseconcentrations in the body. Thus, the system simulates the body'snatural insulin response to blood glucose levels and not only makesefficient use of insulin, but also accounts for other bodily functionsas well since insulin has both metabolic and mitogenic effects. Further,the glycemic control achieved using a closed loop system is achievedwithout requiring any information about the size and timing of a meal,or other factors. The system can rely solely on real time blood glucosemeasurements. The glucose sensor generates a sensor signalrepresentative of blood glucose levels in the body, and provides thesensor signal to the controller. The controller receives the sensorsignal and generates commands that are communicated to the insulindelivery system. The insulin delivery system receives the commands andinfuses insulin into the body in response to the commands. Providedbelow are descriptions of exemplary components of closed loop systemsthat can be used to deliver the super fast-acting insulin compositionsprovided herein. It is understood that one of skill in the art canreadily identify suitable closed loop systems for use herein. Suchsystems have been described in the art, including but not limited to,those described in U.S. Pat. Nos. 5,279,543, 5,569,186, 6,558,351,6,558,345, 6,589,229, 6,669,663, 6,740,072, 7,267,665 and 7,354,420. Theindividual components of the systems also have been described in theart, individually and in the context of a closed loops system for use inachieving glycemic control. It is understood that the examples providedherein are exemplary only, and that other closed loop systems orindividual components can be used to deliver the super fast-actinginsulin compositions provided herein.

Closed loop systems contain a glucose sensor or monitor that functionscontinuously. Such devices can contain needle-type sensors that areinserted under the skin and attached to a small transmitter thatcommunicates glucose data wirelessly by radiofrequency telemetry to asmall receiver. In some examples, the sensor is inserted through thesubject's skin using an insertion needle, which is removed and disposedof once the sensor is positioned in the subcutaneous tissue. Theinsertion needle has a sharpened tip and an open slot to hold the sensorduring insertion into the skin (see e.g. U.S. Pat. Nos. 5,586,553 and5,954,643). The sensor used in the closed loop system can optionallycontain three electrodes that are exposed to the interstitial fluid(ISF) in the subcutaneous tissue. The three electrodes include a workingelectrode, a reference electrode and a counter electrode that are usedto form a circuit. When an appropriate voltage is supplied across theworking electrode and the reference electrode, the ISF providesimpedance between the electrodes. An analog current signal flows fromthe working electrode through the body and to the counter electrode. Thevoltage at the working electrode is generally held to ground, and thevoltage at the reference electrode can be held at a set voltage Vset,such as, for example, between 300 and 700 mV. The most prominentreaction stimulated by the voltage difference between the electrodes isthe reduction of glucose as it first reacts with the glucose oxidaseenzyme (GOX) to generate gluconic acid and hydrogen peroxide (H₂O₂).Then the H₂O₂ is reduced to water (H₂O) and (O⁻) at the surface of theworking electrode. The O⁻ draws a positive charge from the sensorelectrical components, thus repelling an electron and causing anelectrical current flow. This results in the analog current signal beingproportional to the concentration of glucose in the ISF that is incontact with the sensor electrodes (see e.g. U.S. Pat. No. 7,354,420).

In some examples, more than one sensor is used to measure blood glucose.For example, redundant sensors can be used and the subject can benotified when a sensor fails by the telemetered characteristic monitortransmitter electronics. An indicator also can inform the subject ofwhich sensors are still functioning and/or the number of sensors stillfunctioning. In other examples, sensor signals are combined throughaveraging or other means. Further, different types of sensors can beused. For example, an internal glucose sensor and an external glucosesensor can be used to measure blood glucose at the same time.

Glucose sensors that can be used in a closed loop system that deliversthe super fast-acting insulin compositions provided herein are wellknown and can be readily identified and, optionally, further modified,by one of skill in the art. Exemplary internal glucose sensors include,but are not limited to, those described in U.S. Pat. Nos. 5,497,772,5,660,163, 5,791,344, 5,569,186, 6,895,265. Exemplary of a glucosesensor that uses florescence is that described in U.S. Pat. No.6,011,984. Glucose sensor systems also can use other sensingtechnologies, including light beams, conductivity, jet sampling, microdialysis, micro-poration, ultra sonic sampling, reverse iontophoresis,or other method (e.g. U.S. Pat. Nos. 5,433,197 and 5,945,676, andInternational Pat. Pub. WO199929230). In some examples, only the workingelectrode is located in the subcutaneous tissue and in contact with theISF, and the counter and reference electrodes are located external tothe body and in contact with the skin. The counter electrode and thereference electrode can be located on the surface of a monitor housingand can be held to the skin as part of a telemetered characteristicmonitor. In further examples, the counter electrode and the referenceelectrode are held to the skin using other devices, such as running awire to the electrodes and taping the electrodes to the skin,incorporating the electrodes on the underside of a watch touching theskin. Still further, more than one working electrode can be placed intothe subcutaneous tissue for redundancy. Interstitial fluid also can beharvested from the body of a subject and flowed over an external sensorthat is not implanted in the body.

The controller receives input from the glucose sensor. The controller isdesigned to model a pancreatic beta cell (β-cell) and provide commandsto the insulin delivery device to infuse the required amount of insulinfor glycemic control. The controller utilizes software with algorithmsto calculate the required amount of insulin based upon the glucoselevels detected by the glucose sensor. Exemplary algorithms includethose that model the β-cells closely, since algorithms that are designedto minimize glucose excursions in the body, without regard for how muchinsulin is delivered, can cause excessive weight gain, hypertension, andatherosclerosis. Typically, the system is intended to emulate the invivo insulin secretion pattern and to adjust this pattern consistentwith the in vivo β-cell adaptation experienced by normal healthyindividuals. Control algorithms useful for closed loop systems includethose utilized by a proportional-integral-derivative (PID) controller.Proportional derivative controllers and model predictive control (MPC)algorithms also can be used in some systems (Hovorka et al. (2006)Diabetic Med. 23:1-12). Exemplary algorithms include, but are notlimited to, those described Hovorka et al. (Diabetic Med. (2006)23:1-12), Shimoda et al., (Front Med Biol Eng (1997) 8:197-211),Shichiri et al. (Artif. Organs (1998) 22:32-42), Steil et al. (DiabetesTechnol Ther (2003) 5: 953-964), Kaletz et al., (Acta Diabetol. (1999)36:215) and U.S. Pat. Nos. 5,279,543, 5,569,186, 6,558,351, 6,558,345,6,589,229, 6,740,042, 6,669,663, 6,740,072, 7,267,665, 7,354,420 andU.S. Pat. Pub. No. 20070243567.

In one example, a PID controller is utilized in the closed loop system.A PID controller continuously adjusts the insulin infusion by assessingglucose excursions from three viewpoints: the departure from the targetglucose (the proportional component), the area under the curve betweenambient and target glucose (the integral component), and the change inambient glucose (the derivative component). Generally, the in vivoβ-cell response to changes in glucose is characterized by “first” and“second” phase insulin responses. The biphasic insulin response of aβ-cell can be modeled using components of a proportional, plus integral,plus derivative (PID) controller (see e.g. U.S. Pat. No. 7,354,420).

The controller generates commands for the desired insulin delivery.Insulin delivery systems, such as insulin pumps, are known in the artand can be used in the closed loop systems. Exemplary insulin deliverydevices (such as those described above) include, but are not limited to,those described in U.S. Pat. Nos. 4,562,751, 467,840, 4,685,903,4,373,527, 4,573,994, 6,554,798, 6,641,533, 6,744,350, 6,852,104,6,872,200, 6,936,029, 6,979,326, 6,999,854, 7,025,713 and 7,109,878. Theinsulin delivery devices typically contain one or more reservoirs, whichgenerally are disposable, containing an insulin preparation, such as asuper fast-acting insulin composition described herein. The reservoirscan contain more than one insulin, such as, for example, a basal-actinginsulin and a fast-acting insulin, either co-formulated and contained ina single reservoir or contained separately in two or more reservoirs.For use with a super fast-acting insulin composition, the insulindelivery device can contain one reservoir containing a co-formulatedfast-acting insulin and hyaluronan degrading enzyme composition, or cancontain two or more reservoirs, such that the fast-acting insulin andhyaluronan degrading enzyme compositions are contained separately inseparate reservoirs. In such instances, the insulin delivery device candeliver each composition simultaneously or subsequent to each other. Insome examples, the compositions are delivered using an infusion tube anda cannula or needle. In other examples, the infusion device is attacheddirectly to the skin and the compositions flow from the infusion device,through a cannula or needle directly into the body without the use of atube. In further examples, the infusion device is internal to the bodyand an infusion tube optionally can be used to deliver the compositions.Closed loop systems also can contain additional components, including,but not limited to, filters, calibrators and transmitters.

G. METHODS OF ASSESSING ACTIVITY, PHARMACOKINETICS AND PHARMACODYNAMICS

Assays can be used to assess the in vitro and in vivo activities ofinsulin alone or in combination with a hyaluronan degrading enzyme.Included among such assays are those that assess the pharmacokinetic andpharmaocodynamic properties of subcutaneously orintraperitonally-administered insulin, including bioavailability, andtolerability. The biological activity of both insulin and a hyaluronandegrading enzyme also can be assessed using assays well known in theart. Such assays can be used, for example, to determine appropriatedosages of an insulin, such as a fast-acting insulin, and a hyaluronandegrading enzyme, and the frequency of dosing, for treatment.

1. Pharmacokinetics, Pharmacodynamics and Tolerability

Pharmacokinetic (PK), pharmacodynamic (PD) and tolerability studies,such as those described in Example 1, below, can be performed usinganimal models, including pig models such as those described in Examples11 and 12, or can be performed during clinical studies with patients.Animal models include, but are not limited to, mice, rats, rabbits,dogs, guinea pigs and non-human primate models, such as cynomolgusmonkeys or rhesus macaques. In some instances, pharmacokinetic andtolerability studies are performed using healthy animals or humansubjects. In other examples, the studies are performed using animalmodels of diabetes, such as those described below, or in diabetic humansubjects. Exemplary procedures useful for performing these studiesinclude glucose clamp techniques (Brehm et al (2007) in Clin DiabetesRes: Methods and Techniques. Ed Michael Rosen, pp 43-76, Example 1). Inthe hyperinsulinemic euglycemic clamp procedure, exogenous insulin isinfused to create hyperinsulinemic plasma insulin concentrations, whilethe plasma glucose concentration is kept constant at the euglycemiclevel by means of a variable exogenous glucose infusion. The glucoseinfusion rate (GIR) required to maintain constant glucose levels duringthe period of hyperinsulinemia provides a measure of the effect of theinfused insulin on glucose metabolism. The GIR is a reflection of theamount of glucose being used by the body (i.e. more exogenous glucoseneeds to be infused to maintain normal blood glucose levels i.e. between90-110 mg/dL, when the body is using more glucose), and, therefore, theactivity of the administered insulin (i.e. increased insulin activityresults in reduced endogenous glucose output and increased blood glucoseutilization, resulting in an overall decline of blood glucose). Thus,such a procedure, in addition to being used to assess insulin secretionand insulin resistance in a subject, also can be used to safely assessthe pharmacokinetic and pharmacodynamic properties of an insulin, suchas an insulin co-administered with a hyaluronan degrading enzyme.

The pharmacokinetics of subcutaneously or intraperitoneally administeredinsulin can be assessed by measuring the time-concentration profile ofthe insulin and calculating such parameters as the maximum (peak) seruminsulin concentration (C_(max)), the peak time (i.e. when maximum seruminsulin concentration occurs; t_(max)), and area under the curve (i.e.the area under the curve generated by plotting time versus blood insulinconcentration; AUC), for any given time interval followingadministration. The absolute bioavailability of subcutaneouslyadministered insulin is determined by comparing the area under the curveof insulin following subcutaneous delivery (AUC_(sc)) with the AUC ofinsulin following intravenous delivery (AUC_(iv)). Absolutebioavailability (F), can be calculated using the formula:F=[([AUC]_(sc)×dose_(sc))/([AUC]_(iv)×dose_(iv))]×100. The relativebioavailability (F_(rel)) of two treatments via the same route ofadministration, such as, for example, an insulin with or withoutco-administration with a hyaluronan degrading enzyme, also can becalculated, such as in Example 1. For example, the relativebioavailability (F_(rel)) of subcutaneously administered Humalog®insulin lispro co-administered with rHuPH20 and subcutaneouslyadministered Humalog® insulin lispro alone can be calculated{[AUC(Humalog® insulin lispro/rHuPH20)]/[AUC(Humalog® insulin lisproalone]}×100, where each dose of Humalog® insulin lispro is the same, andthe AUC is calculated over the same time interval. The concentration ofinsulin in the plasma following subcutaneous administration can bemeasured using any method known in the art suitable for assessingconcentrations of insulin in samples of blood. Exemplary methodsinclude, but are not limited to, ELISA and RIA.

The pharmacodynamic properties of subcutaneously or intraperitoneallyadministered insulin, can be assessed by measuring such parameters asthe glucose infusion rate (GIR) (mg/kg/min), time to maximal effect(tGIR_(max)) (minutes); the time to late half-maximal effect(tGIR_(late 50%)) (minutes); the time to early half-maximal effect(tGIR_(early 50%)) (minutes); the maximal metabolic effect (GIR_(max))(mg/kg/minute); AUC-GIR_(0-60 min) (g/kg); AUC-GIR_(0-120 min) (g/kg);AUC-GIR_(0-180 min) (g/kg); AUC-GIR_(0-240 min) (g/kg);AUC-GIR_(0-300 min) (g/kg); and the AUC-GIR_(0-360 min) (g/kg).

A range of doses and different dosing frequency of dosing can beadministered in the pharmacokinetic studies to assess the effect ofincreasing or decreasing concentrations of insulin and/or a hyaluronandegrading enzyme in the dose. Pharmacokinetic and pharmacodynamicproperties of subcutaneously or intraperitonally administered insulin,such as bioavailability, also can be assessed with or withoutco-administration of a hyaluronan degrading enzyme. For example, animalsor human subjects can be administered insulin subcutaneously alone or incombination with a hyaluronan degrading enzyme during a hyperinsulinemiceuglycemic clamp procedure. Blood samples can then be taken at varioustime points and the amount of insulin in the serum determined, such asby radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).The glucose infusion rate throughout the procedure also can becalculated. The pharmacokinetic and pharmacodynamic properties ofsubcutaneously administered insulin administered with or without ahyaluronan degrading enzyme can then be determined to assess the effectof co-administration with a hyaluronan degrading on such properties ofany insulin.

Studies to assess safety and tolerability also are known in the art andcan be used herein. Following subcutaneous administration of insulin,with or without co-administration of a hyaluronan degrading enzyme, thedevelopment of any adverse reactions can be monitored. Adverse reactionscan include, but are not limited to, injection site reactions, such asedema or swelling, headache, fever, fatigue, chills, flushing,dizziness, urticaria, wheezing or chest tightness, nausea, vomiting,rigors, back pain, chest pain, muscle cramps, seizures or convulsions,changes in blood pressure and anaphylactic or severe hypersensitivityresponses. Typically, a range of doses and different dosing frequenciesare administered in the safety and tolerability studies to assess theeffect of increasing or decreasing concentrations of insulin and/or ahyaluronan degrading enzyme in the dose.

2. Biological Activity

a. Insulin

The ability of an insulin, such as an insulin analog, to act as atherapeutic agent can be assessed in vitro or in vivo. For example, invitro assays well known in the art can be performed to assess theability an insulin to bind to insulin receptor. In one example, acompetitive binding assay is performed in which human placental cellmembranes are prepared as a source of insulin receptors and incubatedwith radiolabeled human insulin with or without the unlabeled insulinanalog. The amount of bound radiolabeled insulin is then detected todetermine the ability of the insulin analog to compete for binding andthe relative affinity of the insulin analog for the placental insulinreceptor is calculated (see e.g. Weiss et al., (2001) J. Biol. Chem.276:40018-40024). Other sources of insulin receptors, including othercells that naturally or recombinantly express the insulin receptor, alsocan be used in such competitive binding assays (Duttaroy et al., (2005)Diabetes 54:251-258).

The ability of insulin to stimulate glucose uptake or effect any otherof its typical metabolic outcomes can be assessed in vitro. To measureinsulin-stimulated glucose uptake, adipocytes are incubated with labeledglucose, such as 2-deoxy-D-[2,6³-H]glucose or D-[U-¹⁴C]glucose with orwithout insulin. The incorporated radioactivity is then measured todetermine the amount of glucose uptake in the presence or absence ofinsulin (Louveau et al., (2004) J Endocrin. 181:271-280, Duttaroy etal., (2005) Diabetes 54:251-258). When assessing the activity of aninsulin analog, the activity of human insulin also can be assessed andused for comparison. In vitro assays to assess glucose production inH4IIE cells in the presence of insulin also can be performed (Wang etal., (2000) J. Bioche, 275:14717-14721, Duttaroy et al., (2005) Diabetes54:251-258).

In vivo studies using diabetic or healthy animal models or humansubjects also can be performed to assess the therapeutic activity ofinsulin. Insulin can be administered to animal models of diabetes toassess the effects on blood glucose levels, circulating insulin levels,and hemoglobin Alc (HbAlc), for example. Hemoglobin Alc forms whenglucose attaches to hemoglobin, which occurs when blood glucose levelsare elevated. HbAlc levels in a blood sample can be assessed by, forexample, HPLC, ELISA, RIA or other immunoassay, Normal HbAlc values forhealthy subjects are approximately 4.0-6.2 percent. The AmericanDiabetes Association recommends that it should be below 7% (or below 6%in certain persons) for patients with diabetes to help prevent thecomplications from diabetes. Insulin levels can be measured by, forexample, ELISA or RIA. Glucose levels are typically measured using aglucose sensor or analyzer.

Animal models for type I diabetes include the nonobese diabetic (NOD)mouse and the BioBreeding (BB) rat (Atkinson et al., (1999) Nature Med.5:601-604). Animal models for type 2 diabetes include, but are notlimited to, ob/ob mice and db/db mice, which have mutations in theleptin gene or leptin receptor, respectively, KK mice,Nagoya-Shibata-Yasuda (NSY) mice, Zucker diabetic fatty (ZDF) rats andGato-Katazaki (GK) rats (Cefalu (2006) ILAR Journal 47:186-198). Inother examples, healthy animals are used to test the activity of aninsulin, with or without a hyaluronan degrading enzyme.

b. Hyaluronan Degrading Enzymes

The activity of a hyaluronan degrading enzyme can be assessed usingmethods well known in the art. For example, the USP XXII assay forhyaluronidase determines activity indirectly by measuring the amount ofundegraded hyaluronic acid, or hyaluronan, (HA) substrate remainingafter the enzyme is allowed to react with the HA for 30 min at 37° C.(USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention,Inc, Rockville, Md.). A Hyaluronidase Reference Standard (USP) orNational Formulary (NF) Standard Hyaluronidase solution can be used inan assay to ascertain the activity, in units, of any hyaluronidase. Inone example, activity is measured using a microturbidity assay. This isbased on the formation of an insoluble precipitate when hyaluronic acidbinds with serum albumin. The activity is measured by incubatinghyaluronidase with sodium hyaluronate (hyaluronic acid) for a set periodof time (e.g. 10 minutes) and then precipitating the undigested sodiumhyaluronate with the addition of acidified serum albumin. The turbidityof the resulting sample is measured at 640 nm after an additionaldevelopment period. The decrease in turbidity resulting fromhyaluronidase activity on the sodium hyaluronate substrate is a measureof hyaluronidase enzymatic activity. In another example, hyaluronidaseactivity is measured using a microtiter assay in which residualbiotinylated hyaluronic acid is measured following incubation withhyaluronidase (see e.g. Frost and Stern (1997) Anal. Biochem.251:263-269, U.S. Patent Publication No. 20050260186). The free carboxylgroups on the glucuronic acid residues of hyaluronic acid arebiotinylated, and the biotinylated hyaluronic acid substrate iscovalently coupled to a microtiter plate. Following incubation withhyaluronidase, the residual biotinylated hyaluronic acid substrate isdetected using an avidin-peroxidase reaction, and compared to thatobtained following reaction with hyaluronidase standards of knownactivity. Other assays to measure hyaluronidase activity also are knownin the art and can be used in the methods herein (see e.g. Delpech etal., (1995) Anal. Biochem. 229:35-41; Takahashi et al., (2003) Anal.Biochem. 322:257-263).

The ability of a hyaluronan degrading enzyme to act as a spreading ordiffusing agent also can be assessed. For example, trypan blue dye canbe injected subcutaneously with or without a hyaluronan degrading enzymeinto the lateral skin on each side of nude mice. The dye area is thenmeasured, such as with a microcaliper, to determine the ability of thehyaluronan degrading enzyme to act as a spreading agent (U.S. Patent No.20060104968). The effect of co-administration of hyaluronidase withanother agent, such as an insulin, on the pharmacokinetic andpharmacodynamic properties of that agent also can be assessed in vivousing animal model and/or human subjects, such as in the setting of aclinical trial, as discussed above and demonstrated in Example 1, below.The functional activity of a hyaluronan degrading enzyme that is not ahyaluronidase can be compared to a hyaluronidase using any of theseassays. This can be done to determine what a functionally equivalentamount of a hyaluronan degrading enzyme is. For example, the ability ofa hyaluronan degrading enzyme to act as a spreading or diffusing agentcan be assessed by injecting it into the lateral skin of mice withtrypan blue, and the amount required to achieve the same amount ofdiffusion as, for example, 100 units of a Hyaluronidase ReferenceStandard, can be determined. The amount of hyaluronan degrading enzymerequired is, therefore, functionally equivalent to 100 hyaluronidaseunits.

H. THERAPEUTIC USES

The methods described herein can be used for treatment of any conditionfor which a fast-acting insulin is employed. Insulin can be administeredsubcutaneously, in combination with a hyaluronan degrading enzyme, totreat any condition that is amendable to treatment with insulin.Typically, a hyaluronan degrading enzyme is co-administered with afast-acting insulin. This section provides exemplary therapeutic uses offast-acting insulin. The therapeutic uses described below are exemplaryand do not limit the applications of the methods described herein.Therapeutic uses include, but are not limited to, treatment for type 1diabetes mellitus, type 2 diabetes mellitus, gestational diabetes, andfor glycemic control in critically ill patients. For example,fast-acting insulin can be administered in combination with a hyaluronandegrading enzyme subcutaneously in discrete doses, such as via a syringeor insulin pen, prior to a meal as prandial insulin therapy in subjectswith diabetes to achieve glycemic control. Fast-acting insulin also canbe administered subcutaneously or intraperitonally in combination with ahyaluronan degrading enzyme using an insulin pump or in the context of aclosed loop system to continuously control blood glucose levelsthroughout the day and night and/or to control post-prandial glycemicexcursions. It is within the skill of a treating physician to identifysuch diseases or conditions.

As discussed above, particular dosages and treatment protocols aretypically individualized for each subject. If necessary, a particulardosage and duration and treatment protocol can be empirically determinedor extrapolated. For example, exemplary doses of fast-acting insulinwithout a hyaluronan degrading enzyme can be used as a starting point todetermine appropriate dosages for the methods provided herein. Dosagelevels can be determined based on a variety of factors, such as bodyweight of the individual, general health, age, the activity of thespecific compound employed, sex, diet, metabolic activity, blood glucoseconcentrations, time of administration, rate of excretion, drugcombination, the severity and course of the disease, and the patient'sdisposition to the disease and the judgment of the treating physician.In particular, blood glucose levels, such as measured by a blood glucosesensor, can be measured and used to determine the amount of insulin anda hyaluronan degrading enzyme to be administered to achieve glycemiccontrol. Algorithms are known in the art that can be used to determine adose based on the rate of absorption and level of absorption of thesuper fast-acting compositions provided herein, and also based uponblood glucose levels. Dosages of insulin for post-prandial glycemiccontrol also can be calculated or adjusted, for example, by determiningthe carbohydrate content of a meal (Bergenstal et al., (2008) DiabetesCare, Lowe et al., (2008) Diabetes Res. Clin. Pract., Chiesa et al.,(2005) Acta Biomed. 76:44-48).

1.′ Diabetes Mellitus

Diabetes mellitus (or diabetes) is characterized by an impaired glucosemetabolism. Blood glucose is derived from carbohydrates absorbed in thegut and produced in the liver. Increasing blood glucose levels stimulateinsulin release. The postprandial glucose influx can be 20 to 30 timeshigher than the hepatic production of glucose observed between meals.Early phase insulin release, lasting 10 minutes or thereabouts,suppresses hepatic glucose production and precedes a longer (late) phaseof release, which lasts two hours or more and covers mealtimecarbohydrate influx. Between meals, a low continuous insulin level,basal insulin, covers ongoing metabolic requirements, in particular toregulate hepatic glucose output as well as glucose utilization byadipose tissue, muscle tissue and other target sites. Patients withdiabetes present with elevated blood glucose levels (hyperglycemia).Diabetes can be classified into two major groups: type 1 diabetes andtype 2 diabetes. Type 1 diabetes, or insulin dependent diabetes mellitus(IDDM), is characterized by a loss of the insulin-producing β-cell ofthe islets of Langerhans in the pancreas, leading to a deficiency ofinsulin. The primary cause of the β-cell deficiency is T-cell mediatedautoimmunity. Type 2 diabetes, or non-insulin dependent diabetesmellitus (NIDDM), occurs in patients with an impaired β-cell function.These patients have insulin resistance or reduced insulin sensitivity,combined with reduced insulin secretion. Type 2 diabetes may eventuallydevelop into type 1 diabetes. Also included in diabetes is gestationaldiabetes. Patients with diabetes can be administered insulin to bothmaintain basal insulin levels and to prevent glycemic excursions, suchas following a meal.

a. Type 1 Diabetes

Type 1 diabetes is a T-cell dependent autoimmune disease characterizedby infiltration of the islets of Langerhans, the endocrine unit of thepancreas, and destruction of β-cells, leading to a deficiency in insulinproduction and hyperglycemeia. Type 1 diabetes is most commonlydiagnosed in children and young adults but can be diagnosed at any age.Patients with type 1 diabetes can present with, in addition to lowinsulin levels and high blood glucose levels, polyuria, polydispia,polyphagia, blurred vision and fatigue. Patients can be diagnosed bypresenting with fasting plasma glucose levels at or above 126 mg/dL (7.0mmol/l), plasma glucose levels at or above 200 mg/dL (11.1 mmol/l) twohours after a 75 g oral glucose load, such as in a glucose tolerancetest, and/or random plasma glucose levels at or above 200 mg/dL (11.1mmol/l).

The primary treatment for patients with type 1 diabetes isadministration of insulin as replacement therapy, which is typicallyperformed in conjunction with blood glucose monitoring. Withoutsufficient replacement insulin, diabetic ketoacidosis can develop, whichcan result in coma or death. Patients can be administered subcutaneousinjections of fast-acting insulin using, for example, a syringe orinsulin pen, or an insulin pump to maintain appropriate blood glucoselevels throughout the day and also to control post-prandial glucoselevels. In some instances, an insulin pump, including in the context ofa closed loop system, can be used to deliver insulin intraperitoneally.Thus, patients with type 1 diabetes can be administered the superfast-acting insulin composition described herein subcutaneously orintraperitoneally via syringe, insulin pen, or insulin pump, or anyother means useful for delivering insulin, using the methods describedherein to more rapidly control blood glucose and insulin levels.

b. Type 2 Diabetes

Type 2 diabetes is associated with insulin resistance and, in somepopulations, also by insulinopenia (loss of β-cell function). In type 2diabetes, phase 1 release of insulin is absent, and phase 2 release isdelayed and inadequate. The sharp spike of insulin release occurring inhealthy subjects during and following a meal is delayed, prolonged, andinsufficient in amount in patients with type 2 diabetes, resulting inhyperglycemia. Patients with type 2 diabetes can be administered insulinto control blood glucose levels (Mayfield et al (2004) Am Fam Physican70:489-500). This can be done in combination with other treatments andtreatment regimes, including diet, exercise and other anti-diabetictherapies (e.g. sulphonylureas, biguanides, meglitinides,thiazolidinediones and alpha-glucosidase inhibitors). Thus, patientswith type 2 diabetes can be administered the super fast-acting insulincompositions described herein subcutaneously or intraperitoneally viasyringe, insulin pen, or insulin pump, or any other means useful fordelivering insulin, using the methods described herein to more rapidlycontrol blood glucose and insulin levels. As discussed elsewhere herein,administration of super fast-acting insulin compositions to Type 2diabetic patients can, in addition to achieving better glycemic controlcompared to the corresponding fast-acting insulin, reduce the risk ofweight gain and obesity that is often associated with insulin therapy inType 2 diabetic patients.

c. Gestational Diabetes

Pregnant women who have never had diabetes before but who have highblood glucose levels during pregnancy are diagnosed with gestationaldiabetes. This type of diabetes affects approximately 1-14% of allpregnant women, depending upon the population studied (Carr et al.,(1998) Clinical Diabetes 16). While the underlying cause remainsunknown, it appears likely that hormones produced during pregnancyreduce the pregnant woman's sensitivity to insulin. The mechanism ofinsulin resistance is likely a postreceptor defect, since normal insulinbinding by insulin-sensitive cells has been demonstrated. The pancreasreleases 1.5-2.5 times more insulin in order to respond to the resultantincrease in insulin resistance. Patients with normal pancreatic functionare able to meet these demands. Patients with borderline pancreaticfunction have difficulty increasing insulin secretion and consequentlyproduce inadequate levels of insulin. Gestational diabetes thus resultswhen there is delayed or insufficient insulin secretion in the presenceof increasing peripheral insulin resistance.

Patients with gestational diabetes can be administered insulin tocontrol blood glucose level. Thus, patients with gestational diabetescan be administered the super fast-acting insulin compositions describedherein subcutaneously via syringe, insulin pen, insulin pump orartificial pancreas, or any other means, using the methods describedherein to more rapidly control blood glucose and insulin levels.

2. Insulin Therapy for Critically Ill Patients

Hyperglycemia and insulin resistance occurs frequently in medicallyand/or surgically critically ill patients and has been associated withincreased morbidity and mortality in both diabetic and non-diabeticpatients and in patients with traumatic injury, stroke, anoxic braininjury, acute myocardial infarction, post-cardiac surgery, and othercauses of critical illness (McCowen et al. (2001) Crit Clin. Care17:107-124). Critically ill patients with hyperglycemia have beentreated with insulin to control blood glucose levels. Such treatment canreduce morbidity and mortality amongst this group (Van den Berghe et al.(2006) N. Eng. J Med. 354:449-461). Insulin is typically administeredintravenously to the patient, such as by injection with a syringe by amedical practitioner or by infusion using an insulin pump. In someexamples, algorithms and software are used to calculate the dose. Thus,critically ill patients with hyperglycemia can be administered a superfast-acting insulin composition described herein to control bloodglucose levels, thereby alleviating the hyperglycemia and reducingmorbidity and mortality.

I. COMBINATION THERAPIES

Any of the super fast-acting insulin compositions described herein canbe administered in combination with, prior to, intermittently with, orsubsequent to, other therapeutic agents or procedures including, but notlimited to, other biologics and small molecule compounds. For anydisease or condition, including all those exemplified above, for which afast-acting insulin is indicated or has been used and for which otheragents and treatments are available, the super fast-acting insulincompositions can be used in combination therewith. Depending on thedisease or condition to be treated, exemplary combinations include, butare not limited to, combination with anti-diabetic drugs, including, butnot limited to, sulfonylureas, biguanides, meglitinides,thiazolidinediones, alpha-glucosidase inhibitors, peptide analogs,including glucagon-like peptide (GLP) analogs and, gastric inhibitorypeptide (GIP) analogs and DPP-4 inhibitors. In another example, thesuper fast-acting insulin compositions described herein can beadministered in combination with, prior to, intermittently with, orsubsequent to, with one or more other insulins, including fast-actinginsulin, and basal-acting insulins.

J. ARTICLES OF MANUFACTURE AND KITS

Pharmaceutical compounds of the super fast-acting insulin compositions,insulin and/or hyaluronan degrading enzyme compositions provided hereincan be packaged as articles of manufacture containing packagingmaterial, a pharmaceutical composition which is effective forcontrolling blood glucose levels, such as in diabetic or criticallysubjects, and a label that indicates that the super fast-acting insulincompositions, insulin and/or hyaluronan degrading enzyme compositionsare to be used for controlling blood glucose levels.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, for example, U.S. Pat.Nos. 5,323,907, 5,052,558 and 5,033,352, each of which is incorporatedherein in its entirety. Examples of pharmaceutical packaging materialsinclude, but are not limited to, blister packs, bottles, tubes,inhalers, pumps, bags, vials, containers, syringes, bottles, and anypackaging material suitable for a selected formulation and intended modeof administration and treatment. A wide array of formulations of thecompounds and compositions provided herein are contemplated as are avariety of treatments for any hemostatic disease or disorder.

Super fast-acting insulin compositions, insulin and/or hyaluronandegrading enzyme compositions also can be provided as kits. Kits caninclude a pharmaceutical composition described herein and an item foradministration. The kits also can include additional pharmaceuticalcompositions. In one example, the kits can include one or more of thesuper fast-acting insulin compositions, insulin and/or hyaluronandegrading enzyme compositions provided herein and one or more otherinsulin compositions, such as for example, slow acting orintermediate-acting insulins, including crystalline insulins, or anycombination thereof. The super fast-acting insulin compositions, insulinand/or hyaluronan degrading enzyme compositions and/or otherpharmaceutical compositions can be supplied with a device foradministration, such as a syringe, an insulin pen, a pump, or areservoir that is inserted into an insulin pen, a pump or other deliverydevice. The kit can, optionally, include instructions for applicationincluding dosages, dosing regimens and instructions for modes ofadministration. Kits also can include a pharmaceutical compositiondescribed herein and an item for diagnosis. For example, such kits caninclude a glucose monitor or sensor.

The kits for example also can contain a variety of fast-acting insulincompositions, or other insulin composition, including one or morebasal-acting insulins, provided in separate containers and in varyingdosages, whereby the user is afforded the opportunity to select a giveninsulin dosage, such as a prandial dosage, to the specific circumstancesinvolving an actual or anticipated occurrence of hyperglycemia.

K. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Co-Administration of Recombinant Human PH20 (rHuPH20) andFast-Acting Insulin Facilitates Improved Pharmacokinetics andPharmacodynamics

Insulin, including insulin analogs, is administered to subjects withdiabetes mellitus for the control of hyperglycemia. In an effort to moreeffectively replicate normal physiologic prandial insulin releaseobserved in healthy subjects, clinical studies were performed todetermine if co-administration of recombinant Human PH20 (rHuPH20) couldincrease the early absorption rate and the amount of absorption of theadministered fast-acting insulin. Increased absorption could result inthe fast-acting insulin being even faster-acting and, therefore, moreclosely mimicking the endogenous insulin concentration-time profileobserved in healthy subjects. This could provide clinical benefit withrespect to better glycemic control and reduced weight gain in subjectswith diabetes mellitus. The clinical studies were designed to assesssafety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) ofHumulin® R insulin and Humalog® insulin lispro (both being fast-actinginsulins as described herein) administered subcutaneously either aloneor in combination with rHuPH20.

Example 1a Pharmacokinetics and Pharmacodynamics of Humalog® InsulinLispro or Humulin® R Insulin with and without Co-Administration ofrHuPH20 in Healthy (Non-Diabetic) Subjects

A randomized, double-blind, crossover, two-stage, sequential 2-arm studyto assess subcutaneous administration 20 units (U) Humalog® insulinlispro or Humulin® R insulin with and without co-administration ofrHuPH20 was performed. Twenty-five healthy adult male subjects wereenrolled in the study. In stage 1, 12 subjects received a subcutaneousinjection of Humalog® insulin lispro and rHuPH20 and a separatesubcutaneous injection of Humalog® insulin lispro alone. Injections wereusually 7 days apart, with half of the subjects receiving Humalog®insulin lispro and rHuPH20 first, followed by Humalog® insulin lisproalone, and half of the subjects receiving Humalog® insulin lispro alonefirst, then Humalog® insulin lispro and rHuPH20. In stage 2, 13 subjectsreceived a subcutaneous injection of Humulin® R insulin and rHuPH20 anda separate subcutaneous injection of Humulin® R insulin alone.Injections were usually 7 days apart, with approximately half of thesubjects receiving Humulin® R and rHuPH20 first followed by Humulin® Rinsulin alone, and half of the subjects receiving Humulin® R insulinalone first then Humulin® R insulin and rHuPH20.

Approximately 14 hours prior to each injection, each of the subjectsreceived a dinner based on an American Diabetes Association 2000-caloriemeal plan with 60 g carbohydrates. A snack of 30 g carbohydrate also wasprovided. Approximately 6 hours after dinner, the subjects startedfasting (except water) for at least 8 hours before being started on aHyperinsulinemic-Euglycemic Clamp procedure for an 8 hour period.Pre-treatment blood samples were collected and vital signs and weightwere measured before the subjects were injected with Humalog® insulinlispro, Humalog® insulin lispro/rHuPH20, Humulin® R insulin or Humulin®R insulin/rHuPH20 2 hours after the Hyperinsulinemic-Euglycemic Clampprocedure was initiated. Blood samples were collected at prescribedintervals, as described below, and glucose and insulin levels werequantified for a period of 6 hours.

A. Dosing

As described above, 12 subjects were administered 20 U Humalog® insulinlispro and 300 U rHuPH20 in 220 μL, and 20 U Humalog® insulin lispro in200 μL subcutaneously in the lower left abdominal quadrant in the firststage of the study. The Humalog® insulin lispro/rHuPH20 dose wasprepared by first thawing rHuPH20 (1 mg/mL, equivalent to about 120,000U/mL in 10 mM HEPES/130 mM NaCl at pH ˜7.0.) at room temperature for anhour and asceptically aspirating 0.153 cc (equivalent to 18,360 U)rHuPH20 into a 0.3 cc capacity insulin syringe. The 0.153 cc rHuPH20 wasthen transferred slowly into a vial containing 1.17 mL of 150 U/mLHYLENEX (rHuPH20). From this vial, 1.1 mL was aspirated and transferredinto a vial containing about 10.2 mL of 100 U/mL Humalog® insulin lisproaspirated from the vial. Two hundred and twenty microliters of theHumalog® insulin lispro/rHuPH20 mixture was then aspirated using a 0.3cc capacity insulin syringe and used within 4 hours for subcutaneousadministration to a single subject.

Thus, the Humalog® insulin lispro/rHuPH20 mixture that was delivered was220 μL and contained 300 U rHuPH20 (2.5 μg), 20 U Humalog® insulinlispro, 0.02 mg Human Serum Albumin (From the Hylenex formulation(functions to stabilize rHuPH20 against adsorptive losses and also canhave stabilizing properties relative to insulin and/or act as anoxidation scavenger); 3 mg glycerin (from the Humalog® insulin lisproformulation (present as a pH buffer, stabilizer of insulin and/ortonicity modifier); 0.6 mg m-cresol (from the Humalog® insulin lisproformulation (antimicrobial growth preservative present at elevatedconcentrations to stabilize the insulin hexamer conformation); 0.004 mgzinc (from the Humalog® insulin lispro formulation used to stabilize theinsulin hexamer conformation); 0.18 mg NaCl (from the Hylenexformulation and rHuPH20 API, as a tonicity modifier); 0.4 phosphate,sodium dibasic (from the Hylenex formulation, as a pH buffer); 0.017 mgEDTA, disodium (from the Hylenex formulation as a metal chelator withthe potential to bind Zn²⁺ and Ca²⁺ ions); 0.006 mg calcium chloride(from the Hylenex formulation, forms a complex with EDTA and can improvesubcutaneous injection comfort); 0.006 mg HEPES (from rHuPH20 APIformulation, as pH buffer); water (as the solvent) and NaOH and/or HClfor pH adjustment.

In Stage 2, as described above, 13 subjects were administered both 20 UHumulin R insulin and 240 U rHuPH20 in 200 μL and 20 U Humulin® Rinsulin in 200 μL subcutaneously into the lower left abdominal quadrant.The Humulin® R insulin/rHuPH20 dose was prepared by first aspirating 0.3cc (150 U) from a vial of Humulin® R insulin using a 0.3 cc capacityinsulin syringe and transferring it into a vial containing 1.2 mL of1500 U/mL rHuPH20 (formulated as a 10× composition of HYLENEX). Themixture was gently mixed and 0.3 cc of air was removed from the vialbefore 200 μL (containing 20 U Humulin® R insulin and 240 U rHuPH20) wasaspirated using a 0.3 cc capacity insulin syringe. This was used within4 hours for subcutaneous administration to a single subject. The 20 UHumulin® R insulin in 200 μL dose was prepared by subcutaneously using asingle syringe.

Thus, the Humulin® R insulin/rHuPH20 mixture that was delivered was 200μL and contained 240 USP U rHuPH20 (2 μg), 20 U Humulin® R insulin, 0.16mg Human Serum Albumin (from the 10× HYLENEX formulation, functioning tostabilize rHuPH20 against adsorptive losses and also potentially toprovide stabilizing properties relative to insulin and/or act as anoxidation scavenger); 3 mg glycerin (from the Humulin® R formulation,acting as a pH buffer, stabilizer of insulin and/or tonicity modifier);0.4 mg m-cresol (from the Humulin® R formulation, acting as anantimicrobial growth preservative present at elevated concentrations tostabilize the insulin hexamer conformation); 0.34 mg zinc (from theHumulin® R formulation, acting to stabilize the insulin hexamerconformation); 1.36 mg NaCl (from the 10× Hylenex formulation andrHuPH20 API, acting as tonicity modifier); 0.224 phosphate, sodiumdibasic (from the 10× Hylenex formulation, for pH buffer); 0.161 mgEDTA, disodium (from the 10× Hylenex formulation, as metal chelator withthe potential to bind Zn²⁺ and Ca²⁺ ions); 0.048 mg calcium chloride(from the 10× Hylenex formulation, which forms a complex with EDTA andcan improve subcutaneous injection comfort); water (as the solvent) andNaOH and/or HCl for pH adjustment.

B. Hyperinsulinemic-Euglycemic Clamp Procedure

The effect of co-administration of rHuPH20 on pharmacokinetics andpharmacodynamics of subcutaneously administered Humalog® insulin lisproor Humulin® R insulin was assessed by taking blood samples to measureinsulin (i.e. Humalog® insulin lispro or Humulin® R insulin) and glucoselevels. A Hyperinsulinemic-Euglycemic Clamp Procedure was used tomaintain plasma glucose levels between 90-110 mg/dL so that the insulinpreparations could be administered without causing hypoglycemia.

The procedure consisted of initially obtaining the subject's weight andheight and measuring the vital signs after resting in a sitting positionfor 5 minutes. Both arms were placed in heating pads to dilate the veinsand IV catheters were then inserted. A catheter was placed into theantecubital vein of one arm for infusion of Dextrose 20% via twoseparate stop cocks. The other intra-arterial catheter was placed intothe other arm for sampling of arterialized blood for glucosemeasurements. The heating pad can be removed from the glucose infusionsite, but the retrograde catheter site was maintained at 65° C. Aninitial blood sample was obtained to measure baseline glucose 30 minutesbefore injection of the insulin preparations. Blood was sampled 10minutes and 1 minute before injection of Humalog® insulin lispro,Humalog® insulin lispro/rHuPH20, Humulin® R insulin or Humulin® Rinsulin/rHuPH20, then every 3 minutes for the first 60 minutes, every 15minutes from 60 minutes to 3 hours, then every hour thereafter to 6hours. Each subjects' glucose levels were analyzed throughout theprocedure using a YSI 2300 Glucose Analyzer (YSI Inc.) and the glucoseinfusion rate (GIR) was adjusted as necessary to maintain plasma glucosebetween 90-110 mg/dL. Circulating levels of insulin were analyzed usinga radioimmunsorbant assay (RIA) that quantifies levels of Humalog®insulin lispro and Humulin® R insulin (Millipore BioPharma ServicesDivision, St. Charles Mo.).

C. Effect of Co-Administration of rHuPH20 on the Pharmacokinetics ofFast-Acting Insulin

Several parameters were measured to determine the effect ofco-administration of rHuPH20 on the pharmacokinetics of fast-actinginsulin composition Humalog® insulin lispro and Humulin® R insulin.These included the maximum measured insulin concentration during theselected dosing interval (C_(max)); time to C_(max) (t_(max)); and areaunder the concentration vs. time curves (AUC), which was assessed forvarious time intervals.

1. Effect of Co-Administration of rHuPH20 and Humalog® Insulin onInsulin Pharmacokinetics

The insulin concentration for each time interval followingadministration of Humalog® insulin lispro or Humalog® insulinlispro/rHuPH20 was assessed by RIA, and is set forth in Tables 5 and 6,respectively. The AUC for the different time intervals (0 minutes to xminutes; e.g. AUC_(0-3 minutes), AUC_(0-6 minutes), AUC_(0-9 minutes),etc.) also is provided, as is the relative bioavailability (F_(rel)),which is calculated as the [AUC_(0-x), (Humulin® Rinsulin+rHuPH20)]/[AUC_(0-x) (Humulin® R insulin alone]*100. Theincremental slope, which is determined by calculating the change ingeometric mean insulin levels over a time interval, also is presented,as is the average slope change, which is a smoothed average of threevalues of the incremental slope.

TABLE 5 Insulin concentration in the blood following Humalog ® insulinlispro administration Immunoreactive insulin (pmol/L) Time Geo-Incremental Slope (mins) Mean Median SD SE Mean Slope AUC(0-x) Change(avg) (min) (pmol/L) (pmol/L · min) (pmol · min/L) (pmol/L · min) 0 72.464.8 35.2 10.2 65.2 3 78.7 67.8 31.8 9.6 73.2 2.67 208 6 82.3 84.8 24.97.5 79.0 1.93 436 2.85 9 96.9 86.3 36 10.8 90.8 3.94 691 2.72 12 108.0102.0 54.2 16.4 97.7 2.28 973 3.63 15 126.3 110.6 75.5 21.8 111.7 4.671287 5.16 18 160.9 146.1 116 33.5 137.2 8.52 1661 6.71 21 194.0 143.6175.8 50.7 158.1 6.95 2104 8.21 24 233.9 172.0 228.5 66.0 185.6 9.172619 7.25 27 273.4 198.7 296.5 85.6 202.5 5.63 3201 10.1 30 324.9 242.3332.9 96.1 249.0 15.51 3879 13.47 33 388.1 302.3 359.2 108.3 306.8 19.264712 15.43 36 417.0 325.4 343.8 103.6 341.3 11.51 5685 16.68 39 485.4355.2 419.7 121.2 399.1 19.26 6795 12.2 42 495.2 354.7 381.9 110.3 416.65.83 8019 13.93 45 552.6 430.4 408.6 118.0 466.7 16.71 9344 9.11 48553.6 451.4 387.3 111.8 481.1 4.78 10766 9.9 51 576.7 483.9 384.9 111.1505.7 8.2 12246 9.83 54 612.8 504.7 306.3 88.4 555.2 16.5 13837 3.27 57594.1 476.6 400.1 115.5 510.5 −14.9 15436 −0.47 60 551.3 460.1 258.574.6 501.4 −3.03 16954 75 596.4 595.1 214.5 64.7 561.1 3.98 24923 90573.6 556.5 193.1 55.8 541.6 −1.3 33193 105 584.8 575.7 131.4 37.9 571.82.01 41543 120 566.4 558.9 92.2 26.6 559.3 −0.83 50026 135 530.3 536.476.1 22.0 525.4 −2.26 58162 150 533.6 515.7 92.3 26.6 526.6 0.08 66052165 491.6 486.8 96.9 28.0 482.8 −2.92 73623 180 463.1 467.1 93.3 26.9454.2 −1.9 80650 240 348.6 332.3 97.8 28.2 335.7 −1.98 104350 300 261.1255.5 81.6 23.6 248.7 −1.45 121882 360 190.4 181.9 49.5 14.3 184.2 −1.08134867

TABLE 6 Insulin concentration in the blood following Co-Administrationof Humalog ® insulin lispro and rHuPH20 Immunoreactive insulin (pmol/L)Time Geo- Incremental Slope (mins) Mean Median SD SE Mean Slope AUC(0-x) Change (avg) F_(rel) (min) (pmol/L) (pmol/L · min) (pmol · min/L)(pmol/L · min) (%) 0 70.5 66.3 32.8 9.5 64.3 3 97.0 80.7 36.6 11.0 91.59.09 234 113 6 144.7 112.2 92.4 27.8 126.7 11.73 561 9.68 129 9 183.9117.3 156.9 45.3 151.4 8.22 978 11.91 142 12 254.4 161.1 252.9 73.0198.7 15.78 1503 15.21 154 15 354.6 216.8 391.1 112.9 263.6 21.63 219720.13 171 18 442.8 293.2 475.4 137.2 332.5 22.97 3091 28.43 186 21 539.4387.6 400.5 115.6 454.6 40.7 4271 33.51 203 24 651.7 489.4 426.4 123.1565.2 36.86 5801 40.17 221 27 759.8 621.2 390.2 112.6 694.0 42.94 769035.71 240 30 839.7 705.4 414.5 119.7 775.9 27.31 9895 37.81 255 33 958.2791.4 360.4 104.0 905.4 43.17 12417 33.44 263 36 1040.5 890.4 352.4101.7 994.9 29.83 15267 30.15 269 39 1118.0 940.3 445.8 128.7 1047.317.47 18331 18.89 270 42 1138.7 991.1 431.6 124.6 1075.5 9.38 2151520.93 268 45 1239.1 1181.6 408.6 118 1183.3 35.93 24903 12.26 267 481234.0 1128.7 497.1 143.5 1157.6 −8.53 28414 6.46 264 51 1173.8 1124.0326.9 94.4 1133.6 −8.01 31851 −12.3 260 54 1095.6 1070.5 236.6 68.31072.6 −20.34 35160 −57.21 254 57 924.7 961.4 433.0 125.0 642.7 −143.2937733 −16.71 244 60 1055.0 1006.7 363.8 105.0 983.2 113.48 40172 237 75926.2 890.4 218.2 63.0 905.2 −5.2 54335 218 90 818.2 788.9 212.1 61.2793.7 −7.43 67077 202 105 689.7 667.7 175.3 50.6 666.6 −8.47 78029 188120 586.5 571.2 145.3 41.9 569.5 −6.48 87300 175 135 492.5 482.9 124.235.8 478.0 −6.1 95157 164 150 423.0 432.4 127.4 36.8 405.3 −4.85 101781154 165 371.6 363.2 114.8 33.1 354.7 −3.37 107481 146 180 342.0 339.695.4 27.5 329.3 −1.69 112610 140 240 232.4 248.8 83.0 24.0 218.1 −1.85129033 124 300 178.3 146.6 75.0 21.7 164.1 −0.9 140501 115 360 148.7135.1 62.6 18.1 136.6 −0.46 149523 111

The C_(max) (pmol/L), t_(max) (minutes), and AUC₀₋₃₆₀ (min*pmol/L) forHumalog® insulin lispro and Humalog® insulin lispro co-administered withrHuPH20 is provided in Table 7. The AUC for the different time intervalsis provided in Table 8. The results indicate that subjects who receivedthe Humalog® insulin lispro/rHuPH20 dose had greater exposure toHumalog® lispro insulin at early time intervals than those dosed withHumalog® insulin lispro alone. Table 9 provides a summary of specific PKparameters for each dosing sequence (e.g., PK for Humalog® insulinlispro/rHuPH20 administered 1^(st) (1) or 2^(nd) (2) and both (all)),and a statistical summary demonstrating that the dosing sequence did nothave an effect on the observed pharmacokinetics. The statisticalanalysis determined the p-value of the difference in the PK observedusing the different treatment groups (i.e. Humalog® insulin lispro aloneversus Humalog® insulin lispro/rHuPH20), and the difference in the PKobserved using the different dosing sequences (i.e. Humalog® insulinlispro alone first and then the Humalog® insulin lispro/rHuPH20, versusHumalog® insulin lispro/rHuPH20 first and then Humalog® insulin lisproalone). Also provided in the table is the relative bioavalability(F_(rel)) which is calculated as the [AUC₀₋₃₆₀ (Humalog®insulin/rHuPH20)]/[AUC₀₋₃₆₀ (Humalog® insulin alone]*100.

For insulin PK, median t_(max) was reduced by 54% with theco-administration of rHuPH20, from 105 to 48 min (p=0.0006), an effectseen in all 12 subjects. Mean C_(max) increased 87% from 697 pmol/L whensubjects were administered only Humalog® insulin lispro to 1,300 pmol/L(p=0.0003) with co-administration of rHuPH20. AUC_(0-360 min) increased11% from 134,867 to 149,523 min*pmol/L, whereas at earlier timeintervals differences were more pronounced (i.e. AUC_(0-30 min) andAUC_(0-60 min) increased 155% and 140%, respectively). Inter-subjectvariability (SD/Mean) in t_(max) improved from 34% when subjectsreceived Humalog® insulin lispro alone to 17% when subjects receivedHumalog® insulin lispro in combination with rHuPH20. This exampledemonstrates that Humalog® insulin lispro, by coadministration with ahyaluronan degrading enzyme (rHuPH20) was rendered a super fast-actinginsulin as described here.

TABLE 7 Pharmacokinetics of insulin following subcutaneous Humalog ®insulin lispro injection with and without co-administration of rHuPH20C_(max) t_(max) AUC₀₋₃₆₀ Treatment Subject_ID (pmol/L) (min) (min *pmol/L) F_(rel) Humalog ® 1 590 105 136000 Only 2 496 57 126000 3 562105 106000 4 721 90 150000 5 972 54 154000 6 449 150 105000 7 1770 39174000 8 795 75 156000 9 672 120 138000 10 502 120 113000 11 851 105183000 12 631 150 160000 N 12 12 12 Mean 751 98 142000 SD 357 36 25600Median 652 105 144000 Geometric 697 91 139000 Mean CV % 38.8 44 18.8Geometric Mean C_(max) t_(max) AUC₀₋₃₆₀ Subject_ID (pmol/L) (min) (min *pmol/L) F_(rel) (%) Humalog ® 1 1090 54 192000 141 with 2 1310 57 161000128 rHuPH20 3 1640 48 172000 162 4 853 48 146000 97 5 1140 45 130000 846 971 57 139000 132 7 2000 30 152000 87 8 2420 48 186000 119 9 1320 45135000 98 10 930 57 123000 109 11 1590 39 189000 103 12 1080 48 179000112 N 12 12 12 12 Mean 1360 48 159000 114 SD 473 8 24500 23 Median 123048 157000 110 Geometric 1300 47 157000 112 Mean CV % 32.8 19 15.7Geometric Mean

TABLE 8 Time interval AUC on Geometric Mean Insulin Concentrations forHumalog ® insulin lispro alone or Co-Administered with rHuPH20 AUC(min * pmol/L) Time Humalog ® Humalog ® Percentage Interval Only withrHuPH20 Difference^(a) 0-15 1287 2197 70.7 0-21 2104 4271 103.0 0-303879 9895 155.1 0-45 9344 24903 166.5 0-60 16954 40172 136.9 0-75 2492354335 118.0 0-90 33193 67077 102.1 0-120 50026 87300 74.5 0-150 66052101781 54.1 0-180 80650 112610 39.6 0-360 134867 149523 10.8^(a)Percentage Difference: (AUC_(0-x [rHuPH20]) −AUC_(0-x [no rHuPH20]))/(AUC_(0-x [no rHuPH20]))

TABLE 9 Effect of Humalog ® insulin lispro dosing sequence on observedpharmacokinetics. Dosing Treatment sequence C_(max) t_(max) AUC_(all)Humalog ® 1 mean 688 94 140000 SD 172 38 21000 SE 70 16 8600 2 mean 814102 143500 SD 491 37 31600 SE 200 15 12900 All mean 751 98 141800 SD 35736 25700 SE 103 10 7400 Humalog ® 1 mean 1239 48 156800 with SD 456 1127800 rHuPH20 SE 186 4 11400 2 mean 1485 49 160500 SD 498 4 23300 SE 2032 9500 All mean 1362 48 158700 SD 473 8 24500 SE 137 2 7100 TreatmentDifference p- 0.0003 0.0006 0.0760 value Sequence Group Effect p- 0.78890.7783 0.9948 value

2. Effect of Co-Administration of rHuPH20 and Humulin® R Insulin onInsulin Pharmacokinetics

In stage 2, patients received either the Humulin® R insulin/rHuPH20 dosefirst and the Humulin® R insulin alone dose second, or the Humulin® Rinsulin alone dose first and then the Humulin® R insulin/rHuPH20 doseusually 7 days later. The concentration of insulin at each time pointfollowing administration of Humulin® R insulin or Humulin® R insulinco-administered with rHuPH20 is provided in Tables 10 and 11,respectively. The AUC for the different time intervals (i.e. AUC for 0to x minutes (AUC_((0-x))); e.g. AUC_(0-3 minutes), AUC_(0-6 minutes),AUC_(0-9 minutes), etc.) (Tables 10, 11, and 12), as is the relativebioavalability (F_(rel)), which is calculated as the [AUC_(0-x)(Humulin® R insulin/rHuPH20)]/[AUC_(0-x)(Humulin® R insulin alone]*100.The incremental slope, which is determined by calculating the change ingeometric mean insulin levels over a time interval, also is presented,as is the average slope change, which is a smoothed average of fivevalues of the incremental slope.

TABLE 10 Insulin concentration in the blood following Humulin ® Rinsulin administration Immunoreactive insulin (pmol/L) Time Geo-Incremental AUC Slope (mins) Mean Median SD SE Mean Slope (0-x) Change(avg) 0 67.4 59.2 29.8 8.3 62.4 3 62.3 59.5 26.9 7.5 58.3 −1.38 181 670.3 62.8 29.7 8.2 65.4 2.37 366 9 70.1 64.9 26.1 7.2 66.0 0.22 564 0.7512 71.1 66.3 28.8 8.0 66.5 0.15 762 1.72 15 79.2 74.8 32.3 9.0 73.6 2.38973 2.18 18 89.8 86.8 34.2 9.5 84.1 3.47 1209 3.29 21 104.2 103.4 36.510.1 98.1 4.68 1482 4.92 24 123.3 130.5 46.1 13.9 115.3 5.75 1802 6.3927 149.8 143.2 57.6 16.0 140.2 8.3 2186 7.59 30 179.4 171.3 59.6 16.5169.5 9.75 2650 7.45 33 208.9 202.8 69.9 19.4 198.0 9.5 3202 8.02 36223.9 238.6 79.6 22.1 209.9 3.97 3813 7.30 39 248.3 231.6 78.7 21.8235.6 8.57 4482 6.12 42 261.4 265.9 79.7 22.1 249.8 4.74 5210 4.57 45272.3 274.1 78.1 21.7 261.2 3.81 5976 3.90 48 279.8 280.0 87.6 24.3266.6 1.78 6768 3.00 51 278.6 262.5 77.2 21.4 268.4 0.59 7570 3.04 54292.2 255.0 84.3 23.4 280.5 4.06 8394 2.49 57 313.7 278.3 110.6 30.7295.4 4.95 9258 60 316.2 280.3 111.2 30.8 298.6 1.05 10149 75 349.0320.4 132.7 36.8 325.5 1.79 14829 90 358.0 298.9 152.1 42.2 329.5 0.2719741 105 364.8 363.6 128.5 35.6 344.9 1.03 24798 120 372.9 339.6 111.230.8 358.8 0.92 30076 135 400.8 402.8 123.6 34.3 382.6 1.59 35636 150423.1 490.9 165.2 45.8 391.9 0.62 41445 165 423.9 424.9 164.1 45.5 392.60.04 47329 180 412.6 447.9 148.0 41.1 386.2 −0.43 53169 240 336.0 309.990.4 26.1 325.8 −1.01 74528 300 308.6 292.8 77.0 21.4 299.7 −0.43 93292360 242.9 238.7 64.5 17.9 234.5 −1.09 109319

TABLE 11 Insulin concentration in the blood following Co-Administrationof Humulin ® R insulin and rHuPH20 Immunoreactive insulin (pmol/L) TimeGeo- Incremental AUC Slope (mins) Mean Median SD SE Mean Slope (0-x)Change (avg) F_(rel) 0 55.0 56.9 14.6 4.1 53.2 3 94.7 95.6 20.3 5.6 92.513.08 219 121 6 148.3 142.9 40.6 11.3 141.7 16.42 570 156 9 194.2 174.743.7 12.1 189.9 16.07 1067 13.2 189 12 223.1 227.3 66.1 18.3 213.2 7.761672 16.3 219 15 262.2 250.1 75.6 21.0 251.3 12.69 2369 16.67 244 18352.8 331.6 108.1 30.0 336.9 28.54 3251 17.25 269 21 402.0 381.5 96.626.8 391.7 18.27 4344 18.16 293 24 463.7 437.6 126.5 38.1 448.6 18.975605 10.86 311 27 504.5 506.6 135.6 37.6 485.6 12.33 7006 17.39 321 30492.0 477.1 210.5 58.4 414.3 −23.79 8356 17.64 315 33 614.5 620.5 146.840.7 597.8 61.16 9874 14.5 308 36 675.7 676.6 167.1 46.3 656.3 19.5111755 19.13 308 39 690.4 649.8 194.2 53.9 666.1 3.28 13739 21.57 307 42805.2 786.4 244.9 67.9 772.6 35.49 15897 12.53 305 45 772.3 741.5 235.265.2 737.9 −11.57 18162 11.13 304 48 809.8 811.3 204.4 56.7 785.7 15.9520448 10.65 302 51 847.7 822.0 209.2 58.0 823.2 12.5 22861 6.13 302 54854.1 800.2 222.3 61.6 825.9 0.88 25335 5.77 302 57 894.5 840.2 242.667.3 864.5 12.87 27870 301 60 852.3 818.3 229.2 63.6 824.4 −13.37 30404300 75 916.8 937.5 226.5 62.8 890.9 4.44 43268 292 90 835.2 858.4 269.474.7 796.4 −6.3 55923 283 105 774.9 692.0 314.6 87.2 703.0 −6.23 67169271 120 666.1 650.6 248.7 69.0 620.1 −5.53 77092 256 135 599.7 557.3233.7 64.8 550.2 −4.66 85868 241 150 573.9 514.9 185.5 51.5 549.5 −0.0594116 227 165 522.3 452.2 166.8 46.3 500.2 −3.29 101988 215 180 446.2445.7 100.7 27.9 435.6 −4.31 109007 205 240 250.5 255.7 61.2 17.7 243.1−3.21 129369 174 300 172.7 161.0 62.8 17.4 161.3 −1.36 141501 152 360115.5 123.6 36.9 10.2 109.2 −0.87 149614 137

TABLE 12 Time interval AUC on Geometric Mean Insulin Concentrations forHumulin ® R insulin alone or Co-Administered with rHuPH20 AUC (min *pmol/L) Humulin ® R Humulin ® R Percentage Time Interval Only withrHuPH20 Difference^(a) 0-15 973 2369 143.5 0-21 1482 4344 193.1 0-302650 8356 215.3 0-45 5976 18162 203.9 0-60 10149 30404 199.6 0-75 1482943268 191.8 0-90 19741 55923 183.3 0-120 30076 77092 156.3 0-150 4144594116 127.1 0-180 53169 109007 105.0 0-360 109319 149614 36.9^(a)Percentage Difference: (AUC_(0-x [rHuPH20]) −AUC_(0-x [no rHuPH20]))/(AUC_(0-x [no rHuPH20]))

3. Comparison of the Pharmacokinetics of Humalog® Insulin and Humulin® RInsulin with and without Co-Administration of rHuPH20

The pharmacokinetics of Humalog® insulin lispro and Humulin® R insulinwith and without co-administration of rHuPH20 were compared. FIG. 1presents a plot of the geometric mean (for all subjects for eachcomposition) insulin concentrations at each time interval. For bothHumalog® and Humulin® R, the concentration-time curves were shifted up(higher insulin concentrations) and to the left (a faster times). Forexample the geometric mean maximum insulin concentration (C_(max)) wasalmost doubled (to 1200 from 697 pmol/L) for Humalog® and more thandoubled (to 967 from 433 pmol/L) for Humulin® R in the presence ofrHuPH20 relative to control. Similarly, the median time to reach thismaximum concentration (t_(max)) was reduced (from 105 to 48 minutes) forHumalog® and (from 165 to 60 minutes) for Humulin® R in the presence ofrHuPH20 relative to control. This shift to higher concentrations atearlier time points is consistent with an increased rate of absorptionand a constant clearance rate. Thus, co-administration of rHuPH20increased the absorption rate of both Humalog® insulin lispro, afast-acting insulin analog, and Humulin® R insulin, a fast-actingregular insulin.

The natural prandial insulin response includes an immediate bolus thatoccurs over the first 10-15 minutes after eating. This rapid rise ininsulin levels provides an important physiological signal that resultsin shutting down the hepatic glucose release into systemic circulation.Therefore the rise in insulin concentration over 15 minutes is aparticularly important parameter. The data presented above demonstratethat the geometric mean insulin lispro concentrations 15 minutes afteradministration of Humalog® are increased 70% from theirpreadministration levels (from 65 to 112 pmol/L) without rHuPH20, butupon coadministration with rHuPH20, the concentration is more thanquadrupled (from 64 to 264 pmol/L). Even more dramatic, the geometricmean insulin concentration increases only slightly (from 62 to 74pmol/L) for Humulin® R administered without rHuPH20, but are again morethan quadrupled (from 53 to 251 pmol/L) when coadministered withrHuPH20. Thus coadministration with rHuPH20 provides a rapid rise ininsulin concentrations that better represents the early physiologicalprandial insulin response in healthy individuals.

Natural prandial response continues for approximately 2 hours andprovides glycemic control for mealtime carbohydrates, and therefore thecumulative systemic insulin exposure over the first approximately 2hours is another particularly important parameter. According to the dataprovided herein, the cumulative area under the geometric mean insulincurve for the first two hours (AUC₀₋₁₂₀) was increased (from 50,000 to87,000 min*pmol/L) for Humalog® and (from 30,000 to 77,000 min*pmol/L)for Humulin® R in the presence of rHuPH20 relative to control. Similarlythe natural prandial response is effectively complete by about 4 hoursafter a meal, and insulin exposure a lat postprandial times can lead tohypoglycemic excursions. The corresponding exposure from 4 until thelast observations at 6 hours (AUC₂₄₀₋₃₆₀ were reduced (from 31,000 to20,000 min*pmol/L) for Humalog® and (from 35,000 to 20,000 min*pmol/L)for Humulin® R in the presence of rHuPH20 relative to control. Thuscoadministration with rHuPH20 increased the desirable insulin exposureby 175 and 256% and decreased the undesirable insulin exposure by 67 and58%, respectively for coadministration with rHuPH20 relative to control.

Interpatient variability in the pharmacokinetics of insulinadministration require physicians to introduce patients to insulintherapy at subtherapeutic levels and progressively increase the dose toavoid overdosing a patient and risking a hypoglycemic event. Thevariability in pharmacokinetics can be expressed as the coefficient ofvariation (CV; defined as the standard deviation/mean typicallyexpressed as a percentage) for key parameters. The CV of the maximumconcentration (C_(max)) compared between subjects was reduced (from 48%to 35%) for Humalog® and (from 34% to 26%) for Humulin® R in thepresence of rHuPH20 relative to control. The CV of the time to maximumconcentration (t_(max)) was reduced (from 48% to 35%) for Humalog® and(from 32% to 28%) for Humulin® R in the presence of rHuPH20 relative tocontrol. The above data demonstrate that CV of the change in insulinconcentration over the first 15 minutes postadministration was reduced(from 147% to 141%) for Humalog® and (from 165% to 40%) for Humulin® Rin the presence of rHuPH20 relative to control. The CV of the cumulativeinsulin exposure over the first 2 hours (AUC₀₋₁₂₀) was reduced (from 41%to 22%) for Humalog® and (from 34% to 26%) for Humulin® R in thepresence of rHuPH20 relative to control. Thus the interpatientvariability of insulin pharmacokinetics was reduced for insulin whencoadministered with rHuPH20 relative to control.

The pharmacokinetics for Humulin® R insulin were improved byco-administration of rHuPH20 whereby the pharmacokinetics substantiallyresembled the pharmacokinetic profile of Humalog® insulin lispro whenco-administered with rHuPH20. In particular, the rate of insulinabsorption and the serum levels of insulin over the first 20 minuteswere comparable between the two different types of insulin whenco-administered with rHuPH20 (refer to Tables 9 and 13). In contrast,when administered without rHuPH20, Humulin® R insulin exhibits a muchslower rate and decreased level of absorption compared to Humalog®insulin lispro in the early time intervals. Thus, the combination ofrHuPH20, a hyaluronan degrading enzyme, and a fast-acting insulinresults in compositions that act faster and to a greater extent than thefast-acting insulin alone, and, for early times (i.e. less than 20minutes post administration), substantially independent of the type offast-acting insulin.

D. Effect of Co-Administration of rHuPH20 on the Glucose Infusion Rate(GIR) Pharmacodynamics

To assess the pharmacodynamic effect co-administration with rHuPH20 hason the glucose infusion rate (GIR), various pharmacodynamic (orglucodynamic (GD)) parameters were determined for subjects dosed withHumulin® R with and without rHuPH20. These included the time to maximaleffect (tGIR_(max)) (minutes); the time to late half-maximal effect(tGIR_(late 50%)) (minutes); the time to early half-maximal effect(tGIR_(early 50%)) (minutes); the maximal metabolic effect (GIR_(max))(mL/hr); AUC-GIR_(0-60 min); AUC-GIR_(0-120 min); AUC-GIR_(0-180 min);AUC-GIR_(0-240 min); AUC-GIR_(0-300 min); and the AUC-GIR_(0-360 min).GIR was expressed as milliliters of dextrose infused per hour (mL/hr),which can be converted to mg/kg/min using the following:

GIR (mg/kg/min)=[IV infusion rate (mL/hr)×dextrose concentration(g/dL)×0.0167/subjects' mass (kg),

where the dextrose concentration=190.6 mg/mL.

1. Effect of Co-Administration of rHuPH20 and Humalog® Insulin on GIRPharmacodynamics

The glucose infusion rate for each time interval followingadministration of Humalog® insulin lispro alone or Humalog® insulinlispro/rHuPH20 was calculated and is presented in Tables 13 and 14,respectively. Also calculated were the AUC (proportional to thecumulative glucose administration) and the relative AUC (F_(rel)). Theincremental slope, which is determined by calculating the change in GIRover a time interval, also is presented.

TABLE 13 Glucose Infusion Rates Following Humalog ® insulin lisproAdministration GIR (IV Infusion Rate, mL/hr) Incre- mental SlopeAUC(0-x) Time Mean Median SD SE (mL/ (min*mL/ (mins) (mL/hr) hr*min) hr)0 3.1 0 7.4 2.1 3 8.4 0 13.6 3.9 1.78 17 6 9.8 0 16.3 4.7 0.44 45 9 10.80 16.0 4.6 0.36 75 12 10.8 0 16.0 4.6 0 108 15 11.0 0 16.4 4.7 0.06 14118 11.3 0 17.1 4.9 0.08 174 21 14.1 9.0 16.3 4.7 0.94 212 24 15.9 13.015.9 4.6 0.61 257 27 20.9 20.5 19.7 5.7 1.67 312 30 24.3 22.0 20.4 5.91.11 380 33 29.7 29.5 16.2 4.7 1.81 461 36 35.8 37.5 18.0 5.2 2.03 55939 42.0 39.5 20.3 5.9 2.08 676 42 50.1 46.0 27.3 7.9 2.69 814 45 55.748.0 32.9 9.5 1.86 972 48 63.0 55.5 37.2 10.7 2.44 1150 51 68.3 57.542.0 12.1 1.75 1347 54 76.6 69.0 53.2 15.4 2.78 1565 57 85.7 75.5 69.420.0 3.03 1808 60 97.7 82.5 90.0 26.0 4 2083 75 112.3 80.0 77.2 22.30.97 3657 90 130.7 93.0 77.3 22.3 1.23 5479 105 142.3 114.0 73.0 21.10.78 7527 120 155.3 122.0 79.7 23.0 0.86 9759 135 166.4 143.5 76.1 22.00.74 12171 150 170.7 148.0 75.4 21.8 0.28 14699 165 175.8 151.5 74.621.5 0.34 17297 180 178.4 162.5 73.8 21.3 0.18 19954 240 184.9 167.088.3 25.5 0.11 30854 300 141.3 130.0 67.4 19.4 −0.73 40641 360 110.3105.0 50.8 14.7 −0.52 48191

TABLE 14 Glucose Infusion Rates Following Co-Administration of Humalog ®insulin lispro and rHuPH20 GIR (IV Infusion Rate) Incre- mental SlopeAUC(0-x) Time Mean Median SD SE (mL/ (min*mL/ F_(rel) (mins) (mL/hr)hr*min) hr) (%) 0 5.4 0 9 2.6 3 8.8 0 13.5 3.9 1.15 21 124 6 15.8 1018.1 5.2 2.31 58 131 9 11.8 0 14.8 4.3 −1.31 100 132 12 13.6 0 17.2 5.00.58 138 128 15 17.0 10 18.6 5.6 1.14 184 131 18 20.9 25 19.1 5.8 1.3240 138 21 26.3 27 23.6 7.1 1.79 311 147 24 33.8 29.5 27.1 7.8 2.52 401156 27 43.9 40 32.7 9.4 3.36 518 166 30 53.8 49.5 36.2 10.5 3.31 665 17533 68.1 59 43.5 12.6 4.75 847 184 36 82.1 68.5 49.4 14.3 4.67 1073 19239 104.0 80 64.5 18.6 7.31 1352 200 42 115.5 89 64.7 18.7 3.83 1681 20745 127.9 96.5 64.1 18.5 4.14 2046 210 48 134.8 104 66.8 19.3 2.31 2440212 51 142.6 107.5 72.1 20.8 2.58 2856 212 54 145.8 112 70.6 20.4 1.083289 210 57 146.8 121 60.7 17.5 0.31 3728 206 60 159.2 124.5 71.1 20.54.14 4187 201 75 174.6 138.5 84.8 24.5 1.03 6690 183 90 186.3 176 77.022.2 0.78 9397 172 105 182.3 147 78.9 22.8 −0.27 12162 162 120 180.2131.5 83.5 24.1 −0.14 14881 152 135 183.8 132 88.8 25.6 0.24 17611 145150 184.8 139 87.1 25.2 0.06 20375 139 165 185.0 143.5 88.8 25.6 0.0223148 134 180 181.8 139.5 85.1 24.6 −0.22 25899 130 240 139.7 129.5 75.121.7 −0.7 35541 115 300 98.6 85 61.2 17.7 −0.68 42689 105 360 87.7 6562.6 18.1 −0.18 48276 100

The GIR_(max), t_(max), and AUC-GIR for various time intervals also weredetermined for these subjects and are presented in Tables 15 and 16.Table 17 provides a summary of the PD parameters for each dosingsequence (e.g. GIR PD for Humalog® insulin lispro/rHuPH20 administered1^(St) (1) or 2^(nd) (2) and both (all)), and a statistical analysis todetermine whether the dosing sequence affected the observedpharmacodynamics. The statistical analysis determined the p-value of thedifference in the PD observed using the different treatment groups (i.e.Humalog® insulin lispro alone versus Humalog® insulin lispro/rHuPH20),and the difference in the PK observed using the different dosingsequences (i.e. Humalog® insulin lispro alone first and then theHumalog® insulin lispro/rHuPH20, versus Humalog® insulin lispro/rHuPH20first and then Humalog® insulin lispro alone).

TABLE 15 Pharmacodynamics of insulin following subcutaneous Humalog ®insulin lispro injection with and without co-administration of rHuPH2050% tGIR_(early50%) tGIR_(late50%) Treatment Subject_ID GIR_(max)GIR_(max) (min) (min) Humalog ® 1 137 68.5 83 NC Only 2 326 163 98 NC 3247 124 68 NC 4 178 89.0 83 NC 5 119 59.5 68 330 6 158 79.0 98 NC 7 350175 53 270 8 90.0 45.0 47 NC 9 115 57.5 83 330 10 180 90.0 68 330 11 13266.0 56 NC 12 382 191 68 330 N 12 12 12 5 Mean 201 101 72 318 SD 10050.2 17 27 SE 29.0 14.5 5 12 Median 168 84.0 68 330 Geometric 181 90.470 317 Mean CV % 50.5 50.5 24 9 Geometric Mean Humalog ® 1 126 63.0 44270 with 2 320 160 32 270 rHuPH20 3 385 193 44 270 4 200 100 83 360 5149 74.5 50 270 6 260 130 NC NC 7 223 112 26 330 8 109 54.5 29 210 9 15477.0 42 210 10 257 129 38 270 11 138 69.0 38 NC 12 336 168 47 330 N 1212 11 10 Mean 221 111 43 279 SD 91.1 45.6 15 49 SE 26.3 13.2 5 16 Median212 106 42 270 Geometric 205 102 41 275 Mean CV % 43.8 43.8 32 18Geometric Mean NC = not calculated

TABLE 16 Pharmacodynamics of insulin following subcutaneous Humalog ®insulin lispro injection with and without co-administration ofrHuPH20-Interval GIR-AUC. GIR AUC (min*mL/hr) 0-60 0-120 0-180 0-2400-360 Treatment Subject ID GIR_(max) t_(max) min min min min minHumalog ® 1 137 240 1040 5400 13200 21400 33800 2 326 150 3300 1400033100 52200 82800 3 247 240 1770 13500 27100 41400 66500 4 178 240 19408570 18600 29200 46500 5 119 180 1050 5340 11400 18500 28700 6 158 240752 4780 12600 21800 39200 7 350 60 4920 22100 37600 50900 68900 8 90.0135 1490 5390 10500 15500 23100 9 115 165 590 4200 10500 17100 25800 10180 180 2030 9090 18700 29400 44400 11 132 240 2880 8070 14600 2200035600 12 382 240 3240 16700 31600 50800 83000 N 12 12 12 12 12 12 12Mean 201 193 2080 9760 20000 30900 48200 SD 100 58 1280 5640 9800 1420021600 SE 29.0 17 371 1630 2830 4090 6250 Median 168 210 1850 8320 1660025600 41800 Range 292 180 4330 17900 27100 36800 59900 Geometric 181 1811740 8470 18000 28100 44000 Mean CV % Geo- 50.5 42.5 71.9 59.1 50.1 47.346.9 metric Mean Humalog ® 1 126 60 2180 8600 15400 21200 28500 with 2320 135 7800 24500 43400 58800 72300 rHuPH20 3 385 75 5330 25800 4350058100 77000 4 200 90 3020 11400 19300 28100 43200 5 149 165 1990 925017600 24100 31400 6 260 360 2100 9780 16300 22200 36400 7 223 135 659019300 32200 42400 55500 8 109 57 3670 10200 15900 19900 24200 9 154 752250 10300 16500 21300 27300 10 257 150 6070 18800 34000 48100 62600 11138 165 3640 10600 18700 26000 36800 12 336 165 5610 19900 38200 5630084100 N 12 12 12 12 12 12 12 Mean 221 136 4190 14900 25900 35500 48300SD 91.1 82 2010 6350 11400 15900 21200 SE 26.3 24 582 1830 3290 46006120 Median 212 135 3650 11000 19000 27000 40000 Range 276 303 581017200 28100 38900 59800 Geometric 205 118 3750 13700 23800 32500 44200Mean CV % Geo- 43.8 57.5 52.9 42.8 44.6 46.1 46.0 metric Mean

TABLE 17 Effect of Humalog ® insulin lispro dosing sequence on observedpharmacodynamics. GIR AUC (min*mL/hr) Dosing 0-60 0-120 0-180 0-2400-360 Treatment Sequence C_(max) t_(max) min min min min min Humalog ® 1Mean 213 185 1910 9860 20700 32580 51650 alone SD 123 45 1130 5470 1103017460 28930 SE 50 18 460 2230 4500 7130 11810 2 Mean 189 200 2260 967019220 29120 44730 SD 81 73 1510 6340 9380 11300 12810 SE 33 30 620 25903830 4610 5230 all Mean 201 193 2080 9760 19960 30850 48190 SD 100 581280 5640 9790 14140 21630 SE 29 17 370 1630 2830 4080 6250 Humalog ® 1Mean 201 160 3930 13080 22650 31330 43830 and SD 58 105 1950 4720 824011210 12870 rHuPH20 SE 24 43 800 1930 3370 4580 5260 2 Mean 242 112 444016660 29180 39750 52720 SD 118 49 2230 7650 13860 19760 27830 SE 48 20910 3120 5660 8070 11360 all Mean 221 136 4190 14870 25920 35540 48280SD 91 82 2010 6340 11390 15940 21190 SE 26 24 580 1830 3290 4600 6120Treatment Difference 0.3502 0.0627 0.0002 0.0011 0.0044 0.0484 0.9746p-value Sequence Group Effect 0.5517 0.3445 0.9365 0.5879 0.5219 0.50750.5403 p-value

Glucose infusion rate PD data supported the PK findings, showing time tomaximal effect (tGIR_(max)) shortened by 36% when patients wereadministered Humalog® insulin lispro in combination with rHuPH20 (median135 minutes) compared to Humalog® insulin lispro alone (median 210minutes), and maximal metabolic effect (GIR_(max)) increased by 13% froma mean of 181 mL/hr when subjects received Humalog® insulin lispro aloneto 205 mL/hr when subjects received Humalog® insulin lispro and rHuPH20(p=0.35). The time to early half-maximal effect (tGIR_(Early50%)) wasreduced by 38% from a median of 68 when patients were administeredHumalog® insulin lispro alone to 42 min when patients were administeredHumalog® insulin lispro in combination with rHuPH20 (p=0.0006).

2. Effect of Co-Administration of rHuPH20 and Humulin®R Insulin on GIRPharmacodynamics

In stage 2, patients received either the Humulin® R insulin/rHuPH20 dosefirst and the Humulin® R insulin alone dose second, or the Humulin® Rinsulin alone dose first and then the Humulin® R insulin/rHuPH20 doseusually 7 days later. The glucose infusion rate for each time intervalfollowing administration of Humulin® R insulin alone or Humulin® Rinsulin/rHuPH20 was calculated and is presented in Tables 18 and 19,respectively. Also calculated were the AUC and the relative amount ofglucose infused over various times (G_(rel)). The incremental slope,which is determined by calculating the change in GIR over a timeinterval, also is presented.

TABLE 18 Glucose Infusion Rates Following Humulin ® R insulinAdministration GIR Incre- mental Time Slope AUC(0-x) (mins) Mean MedianSD SE (mL/ (min*mL/ (mins) (mL/hr) hr*min) hr) 0 8.5 0 11 3.1 3 15.0 717 4.7 2.17 35 6 15.7 12 17.4 4.8 0.23 81 9 18.0 21 17.8 4.9 0.77 132 1218.8 21 17.8 4.9 0.28 187 15 20.4 21 18.9 5.3 0.51 246 18 20.5 21 19 5.30.05 307 21 22.2 21 18.5 5.1 0.56 371 24 22.9 27 18.2 5 0.23 439 27 24.130 18.5 5.1 0.38 510 30 28.4 30 21 5.8 1.44 588 33 29.3 32 20 5.5 0.31675 36 30.9 32 19.6 5.4 0.54 765 39 32.7 32 19.8 5.5 0.59 861 42 34.8 3420.4 5.7 0.72 962 45 40.2 37 21.6 6 1.77 1075 48 42.2 40 19.4 5.4 0.671198 51 44.3 39 19.3 5.4 0.72 1328 54 47.8 47 17.5 4.8 1.18 1466 57 51.549 17.6 4.9 1.23 1615 60 56.9 63 19.3 5.3 1.79 1778 75 72.5 77 27.4 7.61.04 2749 90 83.6 85 41.7 11.6 0.74 3920 105 92.8 97 47.3 13.1 0.62 5243120 102.6 99 50.1 13.9 0.65 6709 135 119.4 105 55.1 15.3 1.12 8374 150127.5 109 57.2 15.9 0.54 10226 165 138.9 136 55.8 15.5 0.76 12223 180146.2 147 61.5 17.1 0.48 14362 240 178.9 193 61.2 17 0.55 24114 300172.0 176 59.1 16.4 −0.12   34642 360 150.3 164 45.4 12.6 −0:36   44311

TABLE 19 Glucose Infusion Rates Following Humulin ® R insulin andrHuPH20 Administration GIR Incre- mental Slope AUC(0-x) Time Mean MedianSD SE (mL/ (min*mL/ G_(rel) (mins) (mL/hr) hr*min) hr) (%) 0 7.4 0 12.53.5 3 16.0 12 15 4.2 2.86 35 100 6 17.5 19 15.2 4.2 0.51 85 105 9 20.124 15.3 4.3 0.85 142 108 12 21.8 24 14.7 4.1 0.59 205 109 15 24.8 2614.4 4 1 275 112 18 30.6 32 13.2 3.7 1.92 358 116 21 36.4 35 15.7 4.41.92 458 123 24 48.2 45 13.4 3.7 3.95 585 133 27 54.8 47 16.2 4.5 2.21740 145 30 65.9 66 21.6 6 3.69 921 157 33 74.3 74 25.8 7.2 2.79 1132 16836 82.1 78 28.4 7.9 2.59 1366 179 39 91.8 87 28.2 7.8 3.26 1627 189 4299.8 91 33.1 9.2 2.67 1915 199 45 110.5 109 36.8 10.2 3.56 2230 208 48121.5 124 42.4 11.8 3.67 2578 215 51 133.7 134 49.7 13.8 4.05 2961 22354 143.4 145 54.4 15.1 3.23 3377 230 57 153.5 162 62.8 17.4 3.38 3822237 60 164.6 172 73.8 20.5 3.69 4299 242 75 184.8 178 99 27.5 1.34 6920252 90 179.7 194 62.1 17.2 −0.34 9653 246 105 183.9 211 60.5 16.8 0.2812380 236 120 191.1 220 64.1 17.8 0.48 15193 226 135 206.5 216 66 18.31.03 18174 217 150 215.5 206 64 17.8 0.6 21339 209 165 202.9 214 62.417.3 −0.84 24477 200 180 197.4 214 57.1 15.8 −0.37 27479 191 240 181.5183 64.2 17.8 −0.26 38847 161 300 117.5 116 44.6 12.4 −1.07 47819 138360 86.5 80 28.7 8 −0.52 53939 122

3. Comparison of the Pharmacodynamics of Humalog® Insulin Lispro andHumulin® R Insulin with and without Co-Administration of rHuPH20

The pharmacodynamics of Humalog® insulin lispro and Humulin® R insulinwith and without co-administration of rHuPH20 were compared. Therelative effect of co-administration of rHuPH20 on the pharmacodynamicsof each type of insulin was assessed. FIG. 2 presents a plot of theglucose infusion rates at each time interval. It was observed thatco-administration of rHuPH20 and Humalog® or Humulin® R markedly shiftedthe glucose infusion rates as a function of time up and to the leftcompared to when the insulins were administered without rHuPH20, similarto the shift in insulin concentration as a function of time plots. Themaximum infusion rate was increased slightly from a mean of 201 to 221mL/hr for Humalog® and 187 to 203 mL/hr for Humulin® R coadministeredwith rHuPH20 relative to control. Similarly, the time of maximum GIR wasreduced from 193 to 136 minutes for Humalog® and 253 to 206 minutes forHumulin® R coadministered with rHuPH20 relative to control. The onset ofaction, as measured by the time to early half-maximal GIR(tGIR_(early 50%)) was reduced from 72 to 43 minutes for Humalog® and113 to 83 minutes for Humulin® R coadministered with rHuPH20 relative tocontrol.

Mealtime carbohydrates are largely digested and introduced into thesystemic circulation over the first few (e.g. two to four) hours after ameal depending on the type of carbohydrate, and thus the cumulative GIRover the first 2 or 3 hours (e.g. from 0 to 120 minutes) is particularlyrelevant. The cumulative volume of a 190.6 mg/mL glucose solutiondelivered over the first 2 hours increased from 163 to 248 mL forHumalog® and 112 to 226 mL for Humulin® R coadministered with rHuPH20relative to control. Excess glucose metabolism after the mealtimecarbohydrates digestion is complete can lead to adverse hypoglycemicincidents. The cumulative volume of glucose solution delivered from 4 to6 hours decreased from 289 to 212 mL for Humalog® and 337 to 252 mL forHumulin® R coadministered with rHuPH20 relative to control. Thuscoadministration of either a fast-acting insulin analog or a fast-actingregular insulin preparation with rHuPH20 increases the glucose loweringcapacity early to facilitate postprandial digestion and decreases theglucose lowering activity when that activity could lead to hypoglycemicexcursions.

The GIR is a reflection of the amount of glucose being used by the body(i.e. more exogenous glucose needs to be infused to maintain bloodglucose levels between 90-110 mg/dL when the body is using moreglucose), and, therefore, the pharmacological activity of theadministered insulin (i.e. insulin activity results in reducedendogenous glucose output and/or increased blood glucose utilization,resulting in an overall decline of blood glucose). Thus, these datademonstrate that the biological action of each of the insulins wassubstantially increased both in speed (onset of glucose metabolism) andextent when co-administered with rHuPH20, a hyaluronan degrading enzyme,compared to when the insulins were administered without rHuPH20.

In this study, the pharmacodynamic properties of Humulin® R insulin whenco-administered with rHuPH20 were improved whereby the pharmacodynamicssubstantially resembled the pharmacodynamic profile of Humalog® whenco-administered with rHuPH20, in contrast to the substantially delayedpharmacodynamic properties of Humulin® R insulin relative to Humalog®insulin lispro administered in the absence of rHuPH20. The GIR requiredto keep blood glucose levels between 90-110 mg/dL over the first 60minutes, and, by extension, the pharmacological activity of the insulin,particularly in the first 60-90 minutes following injection, wasessentially the same between the two different types of insulin whenco-administered with rHuPH20. In contrast, Humulin® R insulin, which isa fast-acting regular insulin, when administered without rHuPH20 has aGIR profile that indicates a slower rate of insulin action compared toHumalog® insulin lispro insulin when administered without rHuPH20. Thus,the combination of rHuPH20, a hyaluronan degrading enzyme, and afast-acting insulin under, for example, conditions such as thosedescribed in this study results in super fast-acting insulincompositions that act faster and to a greater extent than thefast-acting insulin alone, and, for early times (i.e. less than 60minutes post administration), substantially independent of the type offast-acting insulin.

Example 1b Pharmacokinetics and Postprandial Glycemic Response ofSubcutaneously Injected Humalog® Insulin Lispro or Humulin® R Insulinwith and without Co-Administration of rHuPH20 Following a Liquid Meal inPatients with Type 1 Diabetes Mellitus

A study evaluating the pharmacokinetics (PK) and postprandial glycemicresponse (i.e. the pharmacodynamics (PD)) of subcutaneously injectedHumalog® insulin lispro and Humulin® R insulin, with and withoutco-injection of rHuPH20, following a liquid meal in patients with Type 1Diabetes Mellitus was performed. The study was a single-blind (blindedto patients only), single-center, crossover, liquid meal trial,consisting of a series of standardized liquid meal challenges, in Type 1diabetic patients with 2 hours of pre-dosing and 8 hours of post-dosingblood sampling for PK and PD parameters.

Each subject underwent a series of dose-finding visits for Humalog®insulin lispro and rHuPH20 (Visits 2A-C; up to three injections) todetermine the appropriate individual insulin dose when co-injected withrHuPH20 to cover the liquid meal at optimal glycemic control (defined asmaintaining the patient's postprandial blood glucose within a range of60 mg/dL and 160 mg/dL). Once determined, this same optimized dose wasemployed for a test meal that was covered by Humalog® insulin lisprowithout rHuPH20 (Visit 3). The subjects then underwent the same seriesof investigations (Visits 4A-B; up to two injections) using regularhuman insulin (Humulin® R insulin), to determine the appropriateindividual regular insulin dose with rHuPH20 for optimal glycemiccontrol. The same optimized dose was employed for a test meal that wascovered by Humulin® R insulin without rHuPH20 (Visit 5).

The study allowed comparison of PK profiles and postprandial glucoseexcursions when prandial insulin was administered with or withoutrHuPH20. Postmeal hypoglycemia also was assessed to verify the clinicalrelevance of any observed PK differences. The primary objective was tocompare the early insulin exposure as measured by the primarypharmacokinetic (PK) endpoint of AUC₀₋₆₀ of Humalog® insulin lispro andHumulin® R insulin injected subcutaneously (SC) before a liquid mealwith and without recombinant human hyaluronidase (rHuPH20). Otherinsulin PK parameters measured included C_(max); t_(max); early t_(50%)(time to early half maximal serum concentration), late t_(50%) (time tolate half maximal serum concentration, AUC_(last) (area under theconcentration-time curve from time 0 to the last observation, whichaccording to protocol is 480 minutes postdose); AUC_((0-inf)) (total AUCfrom time 0 to infinity); Interval AUCs (0-15, 0-30, 0-45, 0-60, 0-90,0-120, 0-180, 0-240, 0-360, 0-480, 15-480, 30-480, 45-480, 60-480,90-480, 120-480, 180-480 and 240-480 minutes). λz (terminal eliminationrate constant; determined by linear regression of the terminal points ofthe log-linear serum concentration-time curve); t1/2 (eliminationhalf-life, defined as 0.693/λz); CL/F (clearance as a function ofbioavailability; calculated as Dose/AUC(0-inf)); MRT(last) (meanresidence time from time 0 to the last observation, which according tothe protocol is 480 minutes postdose); MRT(0-inf) (mean residence timefrom time 0 to infinity), and Vz/F volume of distribution as a functionof bioavailability).

Pharmacodynamic (PD) endpoints were postprandial glycemic responseparameters, including AUC_(BG 0-4 h) (where BG denotes blood glucose),and other PD endpoints including AUC_(BG) at specified time intervals,BG_(max), t_(BGmax), early t_(BG 50%), late t_(ag 50%), hypoglycemicepisodes (HE) at specified time intervals, infusion of 20% glucosesolution (amount and duration) to treat hypoglycemia, use of 50% glucosesolution for emergency resuscitation (i.e. presence of severe symptomsand/or blood glucose <36 mg/dL) and hypoglycemic excursions asquantified by AUC above blood glucose 36 mg/dL and below 70 mg/dL.Safety parameters such as adverse events, hematology, biochemistry,urinalyses, physical examinations, vital signs, ECGs, blood glucose,local tolerability at injection site, and antibody formation to insulinagents and to rHuPH20 also were assessed.

A. Patient Selection

Male and female patients with Type 1 diabetes mellitus, treated withinsulin for ≦12 months, were eligible for inclusion in the study. Thepatients were required to be 18 to 65 years old. Females ofchild-bearing potential were required to use a standard and effectivemeans of birth control for the duration of the study. Other inclusioncriteria included: BMI 18.0 to 29.0 kg/m², inclusive; HbAlc(glycosylated hemoglobin Alc)≦10% based on local laboratory results;Fasting C-peptide <0.6 ng/mL; Current treatment with insulin <1.2U/kg/day. Patients also were required to be in good general health basedon medical history and physical examination, without medical conditionsthat might prevent the completion of study drug injections andassessments required in this protocol.

The various study exclusion criteria included: known or suspectedallergy to any component of any of the study drugs in the trial;previous enrollment in the trial; patients with proliferativeretinopathy or maculopathy, and/or severe neuropathy, in particularautonomic neuropathy; clinically significant active disease of thegastrointestinal, cardiovascular (including a history of arrhythmia orconduction delays on ECG), hepatic, neurological, renal, genitourinary,or hematological systems, or uncontrolled hypertension (diastolic bloodpressure ≧100 mmHg and/or systolic blood pressure ≧160 mmHg after 5minutes in the supine position); history of any illness or disease thatmight confound the results of the trial or pose additional risk inadministering the study drugs to the patient; clinically significantfindings in routine laboratory data; anemia with hemoglobin less thanlower limits of normal at screening is specifically exclusionary; use ofdrugs that may interfere with the interpretation of trial results or areknown to cause clinically relevant interference with insulin action,glucose utilization, or recovery from hypoglycemia; recurrent majorhypoglycemia or hypoglycemic unawareness, as judged by the Investigator;current addiction to alcohol or substances of abuse; blood donation(>500 mL) within the previous 9 weeks prior to Visit 2A (see section B,below) on study; pregnancy, breast-feeding, the intention of becomingpregnant, or not using adequate contraceptive measures (adequatecontraceptive measures consist of sterilization, intra-uterine device[IUD], oral or injectable contraceptives or barrier methods); mentalincapacity, unwillingness, or language barriers precluding adequateunderstanding or cooperation; symptomatic gastroparesis; receipt of anyinvestigational drug within 4 weeks of Visit 2A (see section B, below)in this study; any condition (intrinsic or extrinsic) that couldinterfere with trial participation or evaluation of data; current use ofinsulin pump therapy and unwilling to change to Lantus in conjunctionwith a short-acting insulin for the duration of the trial.

Twenty-one evaluable patients completed the trial: 14 male; 7 female;age=41.6±10.6 years; BMI=24.4±286 kg/m²). An evaluable patient was apatient who completed visit 3 and visit 5 and had sufficient bloodsampling and safety assessments for endpoint analyses Any patient whodid not complete all protocol-specified study drug injections and/orwithout sufficient blood sampling and safety assessments through visit 5was replaced by the enrollment of an additional patient.

B. Study Methods

1. Visit Procedures

Each patient attended a screening visit (Visit 1) to determine theeligibility for participation in the trial. Once enrolled, each patienthad at least one and up to three dosing-finding Visits 2A-C (Humalog®insulin lispro with rHuPH20), one dosing Visit 3 (Humalog® insulinlispro alone), at least one and up to two dosing-finding Visits 4A-B(Humulin® R insulin with rHuPH20), one dosing Visit 5 (Humulin® Rinsulin alone), and a follow-up visit (Visit 6).

Patients on an insulin pump, NPH, or any other long acting insulin, whoparticipated in the study, were converted to Lantus for the duration ofthe study. The conversion took place once the subject has passedscreening assessments but at least 36 hours before their first dosingvisit.

Each dose-finding visit and each dosing visit was completed in a singleday. Following early morning check-in, patients were observed andstabilized for approximately 2 hours using intravenous glucose and/orinsulin as required to bring blood glucose into a target value of 100mg/dL. No insulin or glucose infusion was allowed during the 30 minutesimmediately prior to dosing. This was then followed by dosing with thetest article (i.e. Humalog® insulin lispro, Humalog® insulinlispro/rHuPH20, Humulin® insulin or Humulin® insulin/rHuPH20) and thenconsumption of the liquid meal at approximately 8:30 AM. On all dosingvisits, PK and PD assessments proceeded for 8 hours until approximately4:30 PM, at which time the patients received a meal and were dischargedif judged safe.

2. Preparations for the Dose-Finding Visit Procedures

An 18-gauge catheter was inserted in the cubital vein of the same armfor analysis of serum insulin and blood glucose using YSI STAT2300Glucose Analyzer. Blood clotting in the catheter and line was preventedby flushing with 0.15 mmol/L saline. A second 18-gauge PTFE catheter wasplaced in a vein of the opposite forearm for infusion of 20% glucosesolution, saline, and insulin as deemed appropriate during thepre-dosing period. Sixty min prior to dosing, blood glucoseconcentration was determined at the following time-points relative todosing: −60, −30, −20 and −10 min with a YSI STAT2300 Glucose Analyzer.The average of the blood glucose readings from −30, −20 and −10 min wereused to determine the individual patient's fasting blood glucose levelfor each dose-finding and dosing visit. A patient with differencesbetween initial fasting blood glucose values that are deemed too largewas rescheduled for the visit or withdrawn from the study.

3. Pre-Dosing Period

During the run-in period of 2 hours, the blood glucose was monitored asneeded to stabilize blood glucose in the target range. The 2 hour run-inperiod was used to adjust the blood glucose levels as appropriate by IVadministration of glucose and/or insulin by means of a precisioninfusion/syringe pump. No insulin or glucose infusion was administeredduring the 30 minutes immediately prior to dosing. At the time of drugadministration, the blood glucose level of the patient was in a rangebetween 80 and 140 mg/dL (targeting a value as close to the range of100-120 mg/dL as possible)

4. Dosing and Ingestion of Standard Liquid Meal

After the 2-hour run-in period, the study drug injection wasadministered (at timepoint 0) by subcutaneous injection with a syringeinto a lifted skin fold of the abdominal wall. The test articles wereprepared as follows. The Humulin® R insulin only dose was prepared byaspirating the correct dose (as determined at Visit 4) from a vial ofHumulin® R insulin (100 U/mL; Eli Lilly) using a 0.3 cc capacity insulinsyringe. The Humulin® R insulin/rHuPH20 was prepared by first aspirating0.3 cc (150 units) from a vial of Humulin® R insulin (500 U/mL; EliLilly) using a 0.3 cc capacity insulin syringe and transferring it intoa vial containing 1 mL rHuPH20 (20 μg/mL; 3000 U/mL). The solution wasmixed by gentle swirling.

The Humalog® insulin lispro only dose was prepared by aspirating thecorrect dose (as determined at Visit 2) from a vial of Humalog® insulinlispro (100 U/mL; Eli Lilly) using a 0.3 cc capacity insulin syringe.The Humalog® insulin lispro/rHuPH20 was prepared by first thawing a vialof rHuPH20 (1 mg/mL; approximately 1200,000 U/mL) at room temperaturefor 1 to 2 hours. Using a sterile 0.3 cc capacity insulin syringe, 0.27cc of air was drawn into the syringe and expelled in the headspace ofthe rHuPH20 vial, before 0.27 cc (0.27 mg; approximately 32400 U)rHuPH20 was drawn into the syringe. This was then transferred slowly, toprevent foaming, into a vial of Hylenex and gently swirled. Using asterile 3.3 cc insulin syringe, 1.1 mL air was drawn and expelled intothe headspace of the Hylenex (containing an extra 0.27 mg rHuPH20;approximately 32400 U) vial before 1.1 mL of the solution was aspiratedand dispensed into a vial of Humalog® insulin lispro (100 U/mL; EliLilly). The solution was mixed by gentle swirling.

A mean dose of 5.7 (±3.0) Humalog® insulin lispro, with or withoutrHuPH20 (0.2 μg/U insulin) was administered. A mean dose of 6.2 (±3.5)Humulin® R insulin, with or without rHuPH20 (0.2 μg/U insulin) wasadministered. The injection sites for insulins co-administered withrHuPH20 were as follows: injection for Visit 2A was in the leftmid-abdominal region, the next visit (Visit 2B or Visit 3 if Visit 2Bwas not necessary) used the right mid-abdominal region and the nextvisit used the left mid-abdominal region, with subsequent injectionsites alternating accordingly. The injection needle was placed at a 45degree angle and kept in the skin fold for 10 seconds.

Within 10 minutes after study drug dosing, the patients consumed aliquid meal (Ensure) providing 60 gm of carbohydrate. The liquid mealwas fully ingested within 10 minutes. The blood glucose was measured forthe next 8 hours at specified time-points. Additional blood glucosemeasurements for safety purposes were performed as needed.

5. Sampling and Assessment

During the pre-dosing period and following dosing, blood glucoseconcentration was monitored by frequent blood glucose measurements usingthe YSI STAT2300 Glucose Analyzer at the specified timepoints of −60,−30, −20, −10, 0, 3, 6, 9, 12, 15, 20, 25, 30, 40, 45, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 255, 270, 285, 300, 315, 330, 345, 360, 375, 390, 415, 420,430, 445, 460, 475 and 480 minutes. Serial blood samples for thedetermination of serum insulin were drawn at −30, −30, −10, 0, 3, 6, 9,12, 15, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300, 360, 420 and480 minutes.

B. Pharmacokinetics of Humulin® R Insulin and Humalog® Insulin Lisprowith and without rHuPH20

The pharmacokinetics for both Humalog® insulin lispro/rHuPH20 andHumulin® R insulin/rHuPH20 showed accelerated but overall comparableexposure as compared to each without rHuPH20. Table 19a sets forth asummary of various PK parameters for 12 patients. This was an interimanalysis that was performed before data from all patients was collected.Thus, only data from 12 of the 21 patients contributed to this analysis.The effect of co-administration with rHuPH20 is shown by % control,calculated by [mean (geometric or arithmetic)PK value for insulin withrHuPH20]/[mean (geometric or arithmetic)PK value for insulinalone]×100), also is included. Geometric Mean and p-value for logtransformed data for C_(max) and AUC parameters, while based onarithmetic mean and untransformed values for t_(max) and Early & Latet_(50%). The primary endpoint, total insulin exposure over the first 1hour (AUC₀₋₆₀), was increased 135% for Humalog® insulin lispro/rHuPH20compared to Humalog® insulin lispro alone (p=0.0197) and 304% forHumulin® R insulin/rHuPH20 over Humulin® R insulin alone (p=0.0005).Early T_(50%) decreased from 19.9 to 12.6 min (p=0.0002) for Humalog®insulin lispro and 40.1 to 14.8 (p=0.033) for Humulin® R insulin.t_(max) decreased from 43.8 to 27.9 min (p=0.002) for Humalog® insulinlispro and 96.7 to 52.1 (p=0.086) for regular; Late T_(50%) decreasedfrom 98.6 to 68.6 min (p=0.0001) for Humalog® insulin lispro and 219.2to 11 1.2 (p=0.008) Humulin® R insulin.

TABLE 19a Pharmacokinetics of insulin administered with or withoutrHuPH20 in a liquid meal study Humalog ® insulin lispro (N = 12)Humulin ® R insulin (N = 12) Effect of Effect of −rHuPH20 +rHuPH20rHuPH20 −rHuPH20 +rHuPH20 rHuPH20 Median Median % Control Median Median% Control (Range) (Range) (p value)^(a) (Range) (Range) (p value)^(a)Insulin   6   6   6   6 Dose (U)  (3, 16)  (3, 16) (2, 18)  (2, 18)early t_(50%)    20.2    13.6  63%    27.3    16.2  60% (min) (13.3,25.6)  (6.3, 18.2) (p = 0.0002) (14.6, 146.0)  (3.9, 22.9) (p = 0.0329)t_(max)  45  30  67%  60  45  75% (min) (30, 60) (15, 45)  (p = 0.0015)(20, 240)  (20, 150) (p = 0.0856) late t_(50%)    86.6    71.0  82%  172.0   104.5  61% (min)  (69.2, 135.0) (42.5, 93.9)  (p = 0.0001)(91.8, 370.0)  (67.2, 173.0) (p = 0.0066) C_(max)    40.7    53.2 126%   21.1    38.5 186% (pmol/L*U) (25.5, 76.2)  (31.2, 101.5) (p = 0.0394)(6.0, 52.3) (21.7, 76.8)  (p = 0.0047) AUC interval (min*pmol/L*U) 0-601373 2310 135%  583 1495 304%  (947, 3113) (1238, 3683) (p = 0.0197)(150, 1860)  (856, 3600) (p = 0.0005) 0-last 3840 3452 105% 3633 4021133% (1673, 5133) (2133, 6375) (p = 0.7332) (745, 6500) (2417, 5656) (p= 0.1679) 0-inf 4016 3491 102% 3867 4143 105% (1783, 5667) (2167, 6650)(p = 0.9004)  (990, 311467) (2433, 5700) (p = 0.8366) ^(a)Analysis ofvariance using a mixed model with fixed effect for treatment. Anunstructured covariance matrix among repeated measurements, performed onlog-transformed values for AUC and Cmax parameters, and untransformeddata for t_(max) and t_(50%) parameters. Values of 0 were set to 1 priorto log transformation.

Table 19b sets forth a summary of various PK parameters for all of the21 patients that completed the study, showing the mean and standarddeviation. The PK analyses in Table 19b were performed on baselinesubtracted (where baseline was the measurement at time 0) individualHumalog® insulin lispro or Humulin® R insulin concentration versus timedata using the non-compartmental approach (linear trapezoidal rule forAUC calculation). WinNonlin user selection criteria were used in thedetermination of Lambda z, the elimination rate constant, upon whichhalf-life, AUC INF_(obs), MRT, CL, and Vz were based. All measurementslower than 20.0 pM were set to zero for purpose of PK calculation.

The addition of rHuPH20 to Humalog® insulin lispro or Humulin® R insulininjection increased the early insulin exposure. The mean dose-normalizedbaseline subtracted C_(max) was increased 74% from 46.6 to 81.2 pmol/Lwith addition rHuPH20 to Humalog® insulin lispro, and 122% from 25.4 to56.5 pmol/L for Humulin® R insulin. For the primary PK endpoint,AUC_(0-60 min), co-administration with rHuPH20 increased the earlyHumalog® insulin lispro exposure by 75% from 1690 to 2950 min*pmol/L/IUand increased early Humulin® R insulin exposure by 210% from 649 to 2010min*pmol/L/IU relative to control administration without enzyme. Thebioavailability upon coadministration with rHuPH20 was not significantlyaltered relative to control injection of Humalog® insulin lispro alone:98% for AUC_(0-inf) and 116% for AUC_(0-last). The relativebioavailability was 120% for AUC_(0-inf) and 174% for AUC_(0-last) withcoadministration of Humulin® R insulin with rHuPH20 relative to controladministration without enzyme (geometric mean dose-normalized baselinesubtracted data used for these calculations; data not shown).Co-administration of both insulin and lispro with rHuPH20 acceleratedT_(max) and Early and Late T_(50%) compared with control injectionwithout rHuPH20.

The time to peak insulin concentration was faster for Humalog® insulinlispro injection with rHuPH20, with arithmetic mean t_(max) at 38.8minutes, versus 47.1 minutes with Humalog® insulin lispro injectionwithout rHuPH20. Subcutaneous injection of Humulin® R insulin withrHuPH20 resulted in a t_(max) of 58.3 minutes, compared to 104 minuteswithout rHuPH20.

TABLE 19b Pharmacokinetics of insulin administered with or withoutrHuPH20 in a liquid meal study Humalog ® insulin lispro (N = 21)Humulin ® R insulin (N = 21) −rHuPH20 +rHuPH20 −rHuPH20 +rHuPH20 Mean SDMean SD Mean SD Mean SD Cmax 46.6 23.3 81.2 92.9 25.4 13.1 56.5 52.1(pmol/L *U) AUClast 4440 2360 8470 19400 3850 1840 6570 8690 (min*pmol/L*U) AUC0-inf 4680 2580 4410 1700 4200 1620 4810 1580 (min*pmol/L *U)AUC0_15 82.1 105 262 183 36 43.6 188 172 (min*pmol/L *U) AUC0_30 485 3941190 1080 171 125 698 461 (min*pmol/L *U) AUC0_45 1080 681 2190 1980 395269 1340 763 (min*pmol/L *U) AUC0_60 1690 926 2950 2530 649 422 20101070 (min*pmol/L *U) AUC0_90 2680 1320 4050 3800 1210 693 3140 1520(min*pmol/L *U) AUC0_120 3370 1620 4980 6080 1770 934 4050 2320(min*pmol/L *U) AUC0_180 4070 1900 5980 9120 2810 1170 4900 3040(min*pmol/L *U) AUC0_240 4310 2060 7230 14200 3560 1280 5230 3450(min*pmol/L *U) AUC0_360 4500 2440 7950 16900 4190 1540 5950 6040(min*pmol/L *U) AUC0_480 4540 2450 8520 19300 4280 1630 6910 9950(min*pmol/L *U) AUC15_480 4460 2360 8270 19200 4250 1620 6720 9870(min*pmol/L *U) AUC30_480 4050 2140 7330 18300 4110 1610 6210 9690(min*pmol/L *U) AUC45_480 3460 1960 6340 17500 3890 1600 5560 9480(min*pmol/L *U) AUC60_480 2850 1810 5570 16900 3630 1620 4890 9220(min*pmol/L *U) AUC90_480 1860 1490 4470 15600 3080 1600 3770 8820(min*pmol/L *U) AUC120_480 1160 1190 3540 13300 2520 1550 2850 7910(min*pmol/L *U) AUC180_480 473 782 2540 10300 1480 1350 2000 7090(min*pmol/L *U) AUC240_480 223 525 1300 5130 726 903 1680 6620(min*pmol/L *U) Lambda_z 0.0214 0.0094 0.0253 0.00905 0.0224 0.01180.0249 0.0118 (1/min) HL_Lambda_z 38.7 16.3 31.8 14.1 40.8 21.4 36.224.8 (min) t_(max) (min) 47.1 15.2 38.8 40.2 104 65 58.3 32.5 earlyt_(50%) 21.1 5.83 13.9 3.34 38.7 31.6 18.5 10.8 (min) late t_(50%) 11230.5 81.9 45.2 214 70.7 118 30.8 (min) Vz_F_obs (L) 87.4 52.2 67.1 30.2117 141 70.5 50.5 Cl_F_obs (L/min) 1.56 0.668 1.56 0.582 1.81 1.27 1.370.435 MRTlast (min) 86.1 23.3 72.4 34.3 131 50.5 93.6 43.7 MRTINF_obs97.6 31.6 73.6 27 144 38.2 90.7 33.5 (min)C. Comparison of Glycemic Response to Meal Challenge Following RegularHuman Insulin and Insulin Lispro with and without rHuPH20

The glycemic response to a meal challenge was improved Humalog® insulinlispro or Humulin® R insulin was administered with rHuPH20 compared towhen the insulins were administered alone. Table 19c sets forth thepharmacodynamic parameters as measured from 12 patients.Co-administration of either Humalog® insulin lispro or Humulin® Rinsulin with rHuPH20 resulted in reduced postprandial blood glucoselevels relative to control injection without rHuPH20. The maximum bloodglucose observed in the 4 hr postprandial period was reduced from 186 to154 mg/dL when Humalog® insulin lispro was administered with rHuPH20compared to Humalog® insulin lispro alone (p=0.0213) and from 212 to 166mg/dL when Humulin® R insulin was administered with rHuPH20 compared toHumulin® R insulin alone (p=0.0406). 2 hr post prandial glucose (PPG)and total excursion area greater than 140 mg/dL were similarly reduced.The total excursion area less than 70 mg/dL was minimal and similar forall test articles, with a minor trend towards increased area forHumalog® insulin lispro and decreased area for Humulin® R insulin withrHuPH20 co-administration.

TABLE 19c Pharmacodynamics of insulin administered with or withoutrHuPH20 in a liquid meal study Humalog ® insulin lispro (N = 12)Humulin ® R insulin (N = 12) −rHuPH20 +rHuPH20 −rHuPH20 +rHuPH20 MedianMedian % Control Median Median % Control (Range) (Range) (p value)^(a)(Range) (Range) (p value)^(a) BG_(max) 186 154 83% 212 166 79% (mg/dL)(127, 270)  (98, 196) (p = 0.0213) (128, 343) (137, 274)  (p = 0.0406)t_(BGmax)  70  95 136%   90  70 78% (min) (30, 120) (20, 240) (p =0.1854)  (45, 120) (45, 140) (p = 0.5744) 2 hr PPG 156 124 80% 192 13269% (mg/dL) (70, 239) (74, 194) (p = 0.0862) (101, 329) (79, 207) (p =0.0084) AUC > 140 3573  400 11% 5254  847 16% (mg*min/dL)   (0, 15758) (0, 6864) (p = 0.0693)   (0, 35013)   (2, 14513) (p = 0.2105) AUC < 70 0  0  0% 347  0  0% (mg*min/dL) (0, 0)   (0, 642) (p = 0.0958)   (0,1148)  (0, 939) (p = 0.2803) ^(a)t-test, paired, 2-tailed

D. Safety

No serious adverse events (AEs) were reported. The mostcommonly-reported AE was decreased blood glucose/hypoglycemia (147events). Of the 147 events of decreased blood glucose/hypoglycemia, 21were considered possibly or probably related to rHuPH20. 17 events wererated as moderate in intensity, 4 of which were considered possiblyrelated to rHuPH20. The remaining 126 events were rated as mild inintensity. All other AEs occurred with less than 5% frequency in thisstudy.

All episodes of hypoglycemia (defined as having a blood glucose valueof >70 mg/dL) regardless of symptoms were captured as AEs in this study.

E. Summary

Co-administration of either Humalog® insulin lispro or Humulin® Rinsulin with rHuPH20 resulted in earlier insulin exposure with earliert_(max), early t_(50%) and late t_(50%) parameters, as well as greaterpeak insulin concentration relative to control injections withoutrHuPH20, without a significant change in bioavailability. This earlierinsulin exposure led to less postprandial hyperglycemia, with reducedpeak 0-4 hr glucose levels, reduced 2 hr postprandial glucose levels,and less hyperglycemic excursions as measured by AUC >140 mg/dL. Thehypoglycemic excursions, as measured by AUC <70 mg/dL, were minimal andsimilar for all test articles, with a minor trend towards increased areafor Humalog® insulin lispro and decreased area for regular human insulin(Humulin® R insulin) upon rHuPH20 co-administration.

Example 1c Pharmacokinetics and Pharmacodynamics of SubcutaneouslyAdministered Humulin® R Insulin or Humalog® Insulin Lispro with orwithout Varying Doses of Recombinant rHuPH20 in Healthy Human Subjects

As part of a single center, phase I, open-label, single-blind (subjectsblinded to the contents of each injection), 4 stage study to determinethe pharmacokinetics, pharmacodynamics (or glucodynamics; GD), safety,tolerability, and optimal ratio of rHuPH20:insulin, a range of rHuPH20dose ratios were administered subcutaneously (SC) with doses of regularinsulin (Humulin® R insulin) or Humalog® insulin lispro, and thepharmacokinetics (PK) and optimum ratio of rHuPH20:insulin was assessedby determining t_(max), C_(max), AUC_(0→t), and relative bioavailabilitybased on serum insulin concentrations collected at specified timepoints.

The effect of co-administration of varying doses of rHuPH20 onpharmacokinetics and pharmacodynamics (or glucodynamics (GD)) ofsubcutaneously administered Humulin® R insulin or Humalog® insulinlispro was assessed by taking blood samples to measure insulin andglucose levels. A Hyperinsulinemic-Euglycemic Clamp Procedure (asdescribed in Example 1) was used to maintain plasma glucose levelsbetween 90-110 mg/dL. Insulin concentrations were assessed to determinethe insulin PK parameters: t_(max), early t_(50%), late t_(50%),AUC_(0→t) and AUC_(t→end) (where t=30, 60, 90, 120, 180, 240, 360, and480 min after injection), AUC_(0→all), AUC_(0→inf), C_(max), relativebioavailability (with compared to without rHuPH20), and inter- andintra-subject variability based on coefficient of variation for all PKparameters. The glucose infusion rate (GIR) rate to maintain euglycemiawhile on clamp was measured and used to determine the following GDparameters: tGIR_(max), early tGIR_(50%), late tGIR_(50%), GIR AUC_(0→t)and GIR AUC_(t→end) (where t=30, 60, 90, 120, 180, 240, 360, and 480 minafter injection), GIR AUC_(0→all), and cGIR_(max), and inter- andintra-subject variability based on coefficient of variation for all GDparameters. The safety and local tolerability of each of the SCinjections also was assessed.

A. Administration Humulin® R Insulin with or without Varying Doses ofrHuPH20

Healthy volunteers were administered 30 μL or 120 μL of Humulin® Rinsulin (diluted to 100 U/mL) with a final concentration of either 0μg/mL, 1.25 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL or 80 μg/mL rHuPH20(approximately 0 U/mL, 150 U/mL, 600 U/mL, 1200 U/mL, 2400 U/mL or 9600U/mL, respectively). Thus, the volunteers were administered either 30 μLcontaining 3 U Humulin® R insulin with approximately 0, 4.5, 18, 36, 72or 288 Units rHuPH20, or 120 μL containing 12 U Humulin® R insulin withapproximately 0, 18, 72, 144, 288 or 1152 Units rHuPH20. Table 19d setsforth the measured pharmacokinetic parameters for the subjects receiving12 U insulin. The PK parameters characteristic of hyaluronidaseco-administration (earlier t_(max) and t_(1/2max), greater C_(max) andearly systemic exposure e.g. AUC_(0-60 min)) were increased comparablyfor all rHuPH20 concentrations tested compared to when insulin wasadministered alone. Glucose infusion rate (GIR) profiles for all rHuPH20concentrations were different from placebo (i.e. 0 μg/mL) with acharacteristic increase in early rates and decrease in late glucoseinfusion. Over the doses tested, all rHuPH20 concentrations weresimilarly effective, and a non-effective dose was not observed.

TABLE 19d Insulin PK Parameters for 12 U Humulin ® R insulin withvarying doses of rHuPH20 Amount of rHuPH20 Variable 0 1.25 5 10 20 80(Units) Statistic μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL C_(max) N 4 4 4 44 4 (pmol/L) Geo. Mean 192.3 418.1 355.7 323.4 371.0 352.2 CV % 22.933.2 23.7 45.6 32.4 35.5 Median 179.5 432.0 334.0 276.0 353.0 371.0t_(max) N 4 4 4 4 4 4 (minutes) Arith. 121.5 108.8 71.3 93.8 75.0 101.3Mean (125.42) (33.26) (14.36) (39.45) (21.21) (41.31) (std) CV % 10330.6 20.2 42.1 28.3 40.8 Median 90.0 105.0 67.5 82.5 82.5 97.5 Earlyt_(50%) N 4 4 4 4 4 4 (minutes) Arith. 41.3 33.3 22.3 30.4 24.7 33.5Mean (25.67) (9.14) (6.97) (9.58) (3.99) (15.10) (std) CV % 62.2 27.431.2 31.5 16.2 45.1 Median 30.3 33.5 25.4 29.3 25.0 34.2 Late t_(50%) N4 4 4 4 4 4 (minutes) Arith. 359.0 196.3 209.8 194.5 210.8 193.8 Mean(28.28) (47.35) (47.08) (69.25) (50.09) (43.26) (std) CV % 7.9 24.1 22.435.6 23.8 22.3 Median 359.0 201.0 204.0 174.5 231.0 187.0 AUC₀₋₆₀ N 4 44 4 4 4 (min*pmol/L) Geo. Mean 4606.8 11299.9 11679.1 9078.3 12193.69514.5 CV % 51.8 38.6 27.6 63.2 44.1 67.0 Median 4635.0 10475.0 11865.07190.0 11425.0 9885.0 AUC_(last) N 4 4 4 4 4 4 (min*pmol/L) Geo. Mean59362.0 76639.9 75575.6 64666.6 70945.2 65635.2 CV % 20.0 11.2 10.6 18.324.5 22.1 Median 57750.0 76550.0 76450.0 64100.0 68150.0 63100.0B. Administration Humalog® Insulin Lispro with or without Varying Dosesof rHuPH20

Healthy volunteers were administered 30 μL or 120 μL of Humalog® insulinlispro (diluted to 50 U/mL) with a final concentration of either 0μg/mL, 0.078 μg/mL, 0.3 μg/mL, 1.2 μg/mL, 5 μg/mL or 20 μg/mL rHuPH20(approximately 0 U/mL, 9.36 U/mL, 36 U/mL, 144 U/mL, 600 U/mL or 2400U/mL, respectively). Thus, the volunteers were administered either 30 μLcontaining 1.5 U Humalog® insulin lispro with approximately 0, 0.28,1.08, 4.32, 18 or 72 Units rHuPH20, or 120 μL containing 6 U Humalog®insulin lispro with approximately 0, 1.12, 4.32, 17.28, 72 or 288 UnitsrHuPH20. Table 19e sets forth the measured pharmacokinetic parametersfor the subjects receiving 6 U Humalog® insulin lispro. Over the dosestested, all rHuPH20 concentrations greater than 0.3 μg/mL were similarlyeffective.

TABLE 19e Insulin PK Parameters for 6 U Humalog ® insulin lispro withvarying doses of rHuPH20 Amount of rHuPH20 Variable 0 0.08 0.31 1.25 520 (units) Statistic μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL C_(max) N 4 4 44 4 4 (pmol/L) Geo. Mean 381.8 355.9 435.1 483.7 579.5 463.6 CV % 13 2126 23 29 32 Median 385 376 463 506 532 438.8 t_(max) N 4 4 4 4 4 4(minutes) Arith. 67.5 36.3 33.8 41.3 33.8 40.0 Mean (8.7) (10.3) (7.5)(14.4) (7.5) (15.8) (std) CV % 13 28 22 35 22 40 Median 67.5 37.5 3037.5 30 37.5 Early t_(50%) N 4 4 4 4 4 4 (minutes) Arith. 25.9 15.6 15.217.0 16.0 15.6 Mean (2.8) (2.6) (3.2) (1.0) (4.5) (0.8) (std) CV % 11 1721 6 28 5 Median 26.1 16.4 14.8 16.5 15.7 15.8 Late t_(50%) N 4 4 4 4 44 (minutes) Arith. 120.0 85.6 92.8 85.4 80.4 77.1 Mean (10.5) (17.4)(23.5) (20.9) (11.6) (21.8) (std) CV % 9 20 25 25 14 28 Median 120 79.888.0 82.2 83.1 77.2 AUC₀₋₆₀ N 4 4 4 4 4 4 (min*pmol/L) Geo. Mean 1165814886 18299 19494 23424 18523 CV % 18 20 22 17 27 25 Median 11600 1615019400 20050 22150 17650 AUC_(last) N 4 4 4 4 4 4 (min*pmol/L) Geo. Mean38590 30890 41165 39504 47405 36705 CV % 9 10 33 14 17 12 Median 3920030950 36350 38150 4610 35700

Example 2 Generation of a Soluble rHuPH20-Expressing Cell Line

The HZ24 plasmid (set forth in SEQ ID NO:52) was used to transfectChinese Hamster Ovary (CHO cells) (see e.g. U.S. patent application SerNos. 10,795,095, 11/065,716 and 11/238,171). The HZ24 plasmid vector forexpression of soluble rHuPH20 contains a pCI vector backbone (Promega),DNA encoding amino acids 1-482 of human PH20 hyaluronidase (SEQ IDNO:49), an internal ribosomal entry site (IRES) from the ECMV virus(Clontech), and the mouse dihydrofolate reductase (DHFR) gene. The pCIvector backbone also includes DNA encoding the Beta-lactamase resistancegene (AmpR), an f1 origin of replication, a Cytomegalovirusimmediate-early enhancer/promoter region (CMV), a chimeric intron, andan SV40 late polyadenylation signal (SV40). The DNA encoding the solublerHuPH20 construct contains an NheI site and a Kozak consensus sequenceprior to the DNA encoding the methionine at amino acid position 1 of thenative 35 amino acid signal sequence of human PH20, and a stop codonfollowing the DNA encoding the tyrosine corresponding to amino acidposition 482 of the human PH20 hyaluronidase set forth in SEQ ID NO:1),followed by a BamHI restriction site. The constructpCI-PH20-IRES-DHFR-SV40pa (HZ24), therefore, results in a single mRNAspecies driven by the CMV promoter that encodes amino acids 1-482 ofhuman PH20 (set forth in SEQ ID NO:3) and amino acids 1-186 of mousedihydrofolate reductase (set forth in SEQ ID NO:53), separated by theinternal ribosomal entry site (IRES).

Non-transfected DG44 CHO cells growing in GIBCO Modified CD-CHO mediafor DHFR(−) cells, supplemented with 4 mM Glutamine and 18 ml/LPlurionic F68/L (Gibco), were seeded at 0.5×10⁶ cells/ml in a shakerflask in preparation for transfection. Cells were grown at 37° C. in 5%CO₂ in a humidified incubator, shaking at 120 rpm. Exponentially growingnon-transfected DG44 CHO cells were tested for viability prior totransfection.

Sixty million viable cells of the non-transfected DG44 CHO cell culturewere pelleted and resuspended to a density of 2×10⁷ cells in 0.7 mL of2× transfection buffer (2×HeBS: 40 mM HEPES, pH 7.0, 274 mM NaCl, 10 mMKCl, 1.4 mM Na₂HPO₄, 12 mM dextrose). To each aliquot of resuspendedcells, 0.09 mL (250 μg) of the linear HZ24 plasmid (linearized byovernight digestion with Cla I (New England Biolabs) was added, and thecell/DNA solutions were transferred into 0.4 cm gap BTX (Gentronics)electroporation cuvettes at room temperature. A negative controlelectroporation was performed with no plasmid DNA mixed with the cells.The cell/plasmid mixes were electroporated with a capacitor discharge of330 V and 960 μF or at 350 V and 960 μF.

The cells were removed from the cuvettes after electroporation andtransferred into 5 mL of Modified CD-CHO media for DHFR(−) cells,supplemented with 4 mM Glutamine and 18 ml/L Plurionic F68/L (Gibco),and allowed to grow in a well of a 6-well tissue culture plate withoutselection for 2 days at 37° C. in 5% CO₂ in a humidified incubator.

Two days post-electroporation, 0.5 mL of tissue culture media wasremoved from each well and tested for the presence of hyaluronidaseactivity, using the microturbidity assay described in Example 5. Resultsare shown in Table 20.

TABLE 20 Initial Hyaluronidase Activity of HZ24 Transfected DG44 CHOcells at 40 hours post-transfection Activity Dilution Units/mlTransfection 1 1 to 10 0.25 330 V Transfection 2 1 to 10 0.52 350 VNegative 1 to 10 0.015 Control

Cells from Transfection 2 (350V) were collected from the tissue culturewell, counted and diluted to 1×10⁴ to 2×10⁴ viable cells per mL. A 0.1mL aliquot of the cell suspension was transferred to each well of five,96 well round bottom tissue culture plates. One hundred microliters ofCD-CHO media (GIBCO) containing 4 mM GlutaMAX™-1 supplement (GIBCO™,Invitrogen Corporation) and without hypoxanthine and thymidinesupplements were added to the wells containing cells (final volume 0.2mL).

Ten clones were identified from the 5 plates grown without methotrexate(Table 21).

TABLE 21 Hyaluronidase activity of identified clones Relative Plate/WellID Hyaluronidase 1C3 261 2C2 261 3D3 261 3E5 243 3C6 174 2G8 103 1B9 3042D9 273  4D10 302

Six HZ24 clones were expanded in culture and transferred into shakerflasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9, 1E11, and4D10 were plated into 96-well round bottom tissue culture plates using atwo-dimensional infinite dilution strategy in which cells were diluted1:2 down the plate, and 1:3 across the plate, starting at 5000 cells inthe top left hand well. Diluted clones were grown in a background of 500non-transfected DG44 CHO cells per well, to provide necessary growthfactors for the initial days in culture. Ten plates were made persubclone, with 5 plates containing 50 nM methotrexate and 5 plateswithout methotrexate.

Clone 3D3 produced 24 visual subclones (13 from the no methotrexatetreatment, and 11 from the 50 nM methotrexate treatment. Significanthyaluronidase activity was measured in the supernatants from 8 of the 24subclones (>50 Units/mL), and these 8 subclones were expanded into T-25tissue culture flasks. Clones isolated from the methotrexate treatmentprotocol were expanded in the presence of 50 nM methotrexate. Clone3D35M was further expanded in 500 nM methotrexate giving rise to clonesproducing in excess of 1,000 Units/ml in shaker flasks (clone 3D35M; orGen1 3D35M). A master cell bank (MCB) of the 3D35M cells was thenprepared.

Example 3 Determination of Hyaluronidase Activity of Soluble rHuPH20

Hyaluronidase activity of soluble rHuPH20 in samples such as cellcultures, purification fractions and purified solutions was determinedusing a turbidometric assay, which is based on the formation of aninsoluble precipitate when hyaluronic acid binds with serum albumin. Theactivity is measured by incubating soluble rHuPH20 with sodiumhyaluronate (hyaluronic acid) for a set period of time (10 minutes) andthen precipitating the undigested sodium hyaluronate with the additionof acidified serum albumin. The turbidity of the resulting sample ismeasured at 640 nm after a 30 minute development period. The decrease inturbidity resulting from enzyme activity on the sodium hyaluronatesubstrate is a measure of the soluble rHuPH20 hyaluronidase activity.The method is performed using a calibration curve generated withdilutions of a soluble rHuPH20 assay working reference standard, andsample activity measurements are made relative to this calibrationcurve.

Dilutions of the sample were prepared in Enzyme Diluent Solution. TheEnzyme Diluent Solution was prepared by dissolving 33.0±0.05 mg ofhydrolyzed gelatin in 25.0 mL of the 50 mM PIPES Reaction Buffer (140 mMNaCl, 50 mM PIPES, pH 5.5) and 25.0 mL of SWFI, and diluting 0.2 mL of25% Buminate solution into the mixture and vortexing for 30 seconds.This was performed within 2 hours of use and stored on ice until needed.The samples were diluted to an estimated 1-2 U/mL. Generally, themaximum dilution per step did not exceed 1:100 and the initial samplesize for the first dilution was not be less than 20 μl. The minimumsample volumes needed to perform the assay were: In-process Samples,FPLC Fractions: 80 μL; Tissue Culture Supernatants:1 mL; ConcentratedMaterial 80 μL; Purified or Final Step Material: 80 μL. The dilutionswere made in triplicate in a Low Protein Binding 96-well plate, and 30μL of each dilution was transferred to Optilux black/clear bottom plates(BD BioSciences).

Dilutions of known soluble rHuPH20 with a concentration of 2.5 U/mL wereprepared in Enzyme Diluent Solution to generate a standard curve andadded to the Optilux plate in triplicate. The dilutions included 0 U/mL,0.25 U/mL, 0.5 U/mL, 1.0 U/mL, 1.5 U/mL, 2.0 U/mL, and 2.5 U/mL.“Reagent blank” wells that contained 60 μL of Enzyme Diluent Solutionwere included in the plate as a negative control. The plate was thencovered and warmed on a heat block for 5 minutes at 37° C. The cover wasremoved and the plate was shaken for 10 seconds. After shaking, theplate was returned to the heat block and the MULTIDROP 384 LiquidHandling Device was primed with the warm 0.25 mg/mL sodium hyaluronatesolution (prepared by dissolving 100 mg of sodium hyaluronate (LifeCoreBiomedical) in 20.0 mL of SWFI. This was mixed by gently rotating and/orrocking at 2-8° C. for 2-4 hours, or until completely dissolved). Thereaction plate was transferred to the MULTIDROP 384 and the reaction wasinitiated by pressing the start key to dispense 30 μL sodium hyaluronateinto each well. The plate was then removed from the MULTIDROP 384 andshaken for 10 seconds before being transferred to a heat block with theplate cover replaced. The plate was incubated at 37° C. for 10 minutes

The MULTIDROP 384 was prepared to stop the reaction by priming themachine with Serum Working Solution and changing the volume setting to240 μL. (25 mL of Serum Stock Solution [1 volume of Horse Serum (Sigma)was diluted with 9 volumes of 500 mM Acetate Buffer Solution and the pHwas adjusted to 3.1 with hydrochloric acid] in 75 mL of 500 mM AcetateBuffer Solution). The plate was removed from the heat block and placedonto the MULTIDROP 384 and 240 μL of serum Working Solutions wasdispensed into the wells. The plate was removed and shaken on a platereader for 10 seconds. After a further 15 minutes, the turbidity of thesamples was measured at 640 nm and the hyaluronidase activity (in U/mL)of each sample was determined by fitting to the standard curve.

Specific activity (Units/mg) was calculated by dividing thehyaluronidase activity (U/ml) by the protein concentration (mg/mL).

Example 4 Production and Purification of Gen1 Human sPH20 A. 5 LBioreactor Process

A vial of 3D35M was thawed and expanded from shaker flasks through 1 Lspinner flasks in CD-CHO media (Invitrogen, Carlsbad Calif.)supplemented with 100 nM Methotrexate and GlutaMAX™-1 (Invitrogen).Cells were transferred from spinner flasks to a 5 L bioreactor (Braun)at an inoculation density of 4×10⁵ viable cells per ml. Parameters weretemperature Setpoint 37° C., pH 7.2 (starting Setpoint), with DissolvedOxygen Setpoint 25% and an air overlay of 0-100 cc/min. At 168 hrs, 250ml of Feed #1 Medium (CD CHO with 50 g/L Glucose) was added. At 216hours, 250 ml of Feed #2 Medium (CD CHO with 50 g/L Glucose and 10 mMSodium Butyrate) was added, and at 264 hours 250 ml of Feed #2 Mediumwas added. This process resulted in a final productivity of 1600 Unitsper ml with a maximal cell density of 6×10⁶ cells/ml. The addition ofsodium butyrate was to dramatically enhance the production of solublerHuPH20 in the final stages of production.

Conditioned media from the 3D35M clone was clarified by depth filtrationand tangential flow diafiltration into 10 mM HEPES pH 7.0. SolublerHuPH20 was then purified by sequential chromatography on Q Sepharose(Pharmacia) ion exchange, Phenyl Sepharose (Pharmacia) hydrophobicinteraction chromatography, phenyl boronate (Prometics) andHydroxapatite Chromatography (Biorad, Richmond, Calif.).

Soluble rHuPH20 bound to Q Sepharose and eluted at 400 mM NaCl in thesame buffer. The eluate was diluted with 2M ammonium sulfate to a finalconcentration of 500 mM ammonium sulfate and passed through a PhenylSepharose (low sub) column, followed by binding under the sameconditions to a phenyl boronate resin. The soluble rHuPH20 was elutedfrom the phenyl sepharose resin in HEPES pH 6.9 after washing at pH 9.0in 50 mM bicine without ammonium sulfate. The eluate was loaded onto aceramic hydroxyapatite resin at pH 6.9 in 5 mM potassium phosphate and 1mM CaCl₂ and eluted with 80 mM potassium phosphate, pH 7.4 with 0.1 mMCaCl₂.

The resultant purified soluble rHuPH20 possessed a specific activity inexcess of 65,000 USP Units/mg protein by way of the microturbidity assay(Example 3) using the USP reference standard. Purified sPH20 eluted as asingle peak from 24 to 26 minutes from a Pharmacia 5RPC styrenedivinylbenzene column with a gradient between 0.1% TFA/H₂O and 0.1%TFA/90% acetonitrile/10% H₂0 and resolved as a single broad 61 kDa bandby SDS electrophoresis that reduced to a sharp 51 kDa band upontreatment with PNGASE-F. N-terminal amino acid sequencing revealed thatthe leader peptide had been efficiently removed.

B. Upstream Cell Culture Expansion Process into 100 L Biorcactor CellCulture

A scaled-up process was used to separately purify soluble rHuPH20 fromfour different vials of 3D35M cell to produce 4 separate batches ofsHuPH20; HUA0406C, HUA0410C, HUA0415C and HUA0420C. Each vial wasseparately expanded and cultured through a 125 L bioreactor, thenpurified using column chromatography. Samples were taken throughout theprocess to assess such parameters as enzyme yield. The description ofthe process provided below sets forth representative specifications forsuch things as bioreactor starting and feed media volumes, transfer celldensities, and wash and elution volumes. The exact numbers vary slightlywith each batch, and are detailed in Tables 24 to 30.

Four vials of 3D35M cells were thawed in a 37° C. water bath, CD CHOcontaining 100 nM methotrexate and 40 mL/L GlutaMAX was added and thecells were centrifuged. The cells were re-suspended in a 125 mL shakeflask with 20 mL of fresh media and placed in a 37° C., 7% CO₂incubator. The cells were expanded up to 40 mL in the 125 mL shakeflask. When the cell density reached 1.5-2.5×10⁶ cells/mL, the culturewas expanded into a 125 mL spinner flask in a 100 mL culture volume. Theflask was incubated at 37° C., 7% CO₂. When the cell density reached1.5-2.5×10⁶ cells/mL, the culture was expanded into a 250 mL spinnerflask in 200 mL culture volume, and the flask was incubated at 37° C.,7% CO₂. When the cell density reached 1.5-2.5×10⁶ cells/mL, the culturewas expanded into a 1 L spinner flask in 800 mL culture volume andincubated at 37° C., 7% CO₂. When the cell density reached 1.5-2.5×10⁶cells/mL, the culture was expanded into a 6 L spinner flask in 5 Lculture volume and incubated at 37° C., 7% CO₂. When the cell densityreached 1.5-2.5×10⁶ cells/mL, the culture was expanded into a 36 Lspinner flask in 20 L culture volume and incubated at 37° C., 7% CO₂.

A 125 L reactor was sterilized with steam at 121° C., 20 PSI and 65 L ofCD CHO media was added. Before use, the reactor was checked forcontamination. When the cell density in the 36 L spinner flasks reached1.8-2.5×10⁶ cells/mL, 20 L cell culture were transferred from the 36 Lspinner flasks to the 125 L bioreactor (Braun), resulting a final volumeof 85 L and a seeding density of approximately 4×10⁵ cells/mL.Parameters were temperature setpoint, 37° C.; pH: 7.2; Dissolved oxygen:25%±10%; Impeller Speed 50 rpm; Vessel Pressure 3 psi; Air Sparge 1L/min.; Air Overlay: 1 L/min. The reactor was sampled daily for cellcounts, pH verification, media analysis, protein production andretention. Nutrient feeds were added during the run. At Day 6, 3.4 L ofFeed #1 Medium (CD CHO+50 g/L Glucose+40 mL/L GlutaMAX™-1) was added,and culture temperature was changed to 36.5° C. At day 9, 3.5 L of Feed#2 (CD CHO+50 g/L Glucose+40 mL/L GlutaMAX™-1+1.1 g/L Sodium Butyrate)was added, and culture temperature was changed to 36° C. At day 11, 3.7L of Feed #3 (CD CHO+50 g/L Glucose+40 mL/L GlutaMAX™-1+1.1 g/L SodiumButyrate) was added, and the culture temperature was changed to 35.5° C.The reactor was harvested at 14 days or when the viability of the cellsdropped below 50%. The process resulted in production of soluble rHuPH20with an enzymatic activity of 1600 Units/ml with a maximal cell densityof 8 million cells/mL. At harvest, the culture was sampled formycoplasma, bioburden, endotoxin, and virus in vitro and in vivo,transmission electron microscopy (TEM) for viral particles, and enzymeactivity.

The one hundred liter bioreactor cell culture harvest was filteredthrough a series of disposable capsule filters having a polyethersulfonemedium (Sartorius): first through a 8.0 μm depth capsule, a 0.65 μmdepth capsule, a 0.22 μm capsule, and finally through a 0.22 μmSartopore 2000 cm² filter and into a 100 L sterile storage bag. Theculture was concentrated 10× using two TFF with Spiral Polyethersulfone30 kDa MWCO filters (Millipore), followed by a 6× buffer exchange with10 mM HEPES, 25 mM Na₂SO₄, pH 7.0 into a 0.22 μm final filter into a 20L sterile storage bag. Table 22 provides monitoring data related to thecell culture, harvest, concentration and buffer exchange steps.

TABLE 22 Monitoring data for cell culture, harvest, concentration andbuffer exchange steps. Parameter HUA0406C HUA04010C HUA0415C HUA0420CTime from thaw to inoculate 100 L 21 19 17 18 bioreactor (days) 100 Linoculation density (×10⁶ cells/mL) 0.45 0.33 0.44 0.46 Doubling time inlogarithmic 29.8 27.3 29.2 23.5 growth (hr) Max. cell density (×10⁶cells/mL) 5.65 8.70 6.07 9.70 Harvest viability (%) 41 48 41 41 Harvesttiter (U/ml) 1964 1670 991 1319 Time in 100-L bioreactor (days) 13 13 1213 Clarified harvest volume (mL) 81800 93300 91800 89100 Clarifiedharvest enzyme assay 2385 1768 1039 1425 (U/mL) Concentrate enzyme assay22954 17091 8561 17785 (U/mL) Buffer exchanged concentrate 15829 116499915 8679 enzyme assay (U/mL) Filtered buffer exchanged 21550 10882 94718527 concentrate enzyme assay (U/mL) Buffer exchanged concentrate 1069913578 12727 20500 volume (mL) Ratio enzyme units 0.87 0.96 1.32 1.4concentration/harvest

A Q Sepharose (Pharmacia) ion exchange column (3 L resin, Height=20 cm,Diameter=14 cm) was prepared. Wash samples were collected for adetermination of pH, conductivity and endotoxin (LAL) assay. The columnwas equilibrated with 5 column volumes of 10 mM Tris, 20 mM Na₂SO₄, pH7.5. The concentrated, diafiltered harvest was loaded onto the Q columnat a flow rate of 100 cm/hr. The column was washed with 5 column volumesof 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 and 10 mM HEPES, 50 mM NaCl, pH 7.0.The protein was eluted with 10 mM HEPES, 400 mM NaCl, pH 7.0 andfiltered through a 0.22 μm final filter into a sterile bag.

Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography wasnext performed. A Phenyl-Sepharose (PS) column (9.1 L resin, Height=29cm, Diameter=20 cm) was prepared. The column was equilibrated with 5column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate, 0.1mM CaCl₂, pH 7.0. The protein eluate from above was supplemented with 2Mammonium sulfate, 1 M potassium phosphate and 1 M CaCl₂ stock solutionsto final concentrations of 5 mM, 0.5 M and 0.1 mM, respectively. Theprotein was loaded onto the PS column at a flow rate of 100 cm/hr. 5 mMpotassium phosphate, 0.5 M ammonium sulfate and 0.1 mM CaCl₂ pH 7.0 wasadded at 100 cm/hr. The flow through was passed through a 0.22 μm finalfilter into a sterile bag.

The PS-purified protein was the loaded onto an aminophenyl boronatecolumn (ProMedics) (6.3 L resin, Height=20 cm, Diameter=20 cm) that hadbeen equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5M ammonium sulfate. The protein was passed through the column at a flowrate of 100 cm/hr, and the column was washed with 5 mM potassiumphosphate, 0.5 M ammonium sulfate, pH 7.0. The column was then washedwith 20 mM bicine, 100 mM NaCl, pH 9.0 and the protein eluted with 50 mMHEPES, 100 mM NaCl pH 6.9 through a sterile filter and into a 20 Lsterile bag. The eluate was tested for bioburden, protein concentrationand enzyme activity.

A hydroxyapatite (HAP) column (BioRad) (1.6 L resin, Height=10 cm,Diameter=14 cm) was equilibrated with 5 mM potassium phosphate, 100 mMNaCl, 0.1 mM CaCl₂ pH 7.0. Wash samples were collected and tested forpH, conductivity and endotoxin (LAL assay. The aminophenyl boronatepurified protein was supplemented with potassium phosphate and CaCl₂ toyield final concentrations of 5 mM potassium phosphate and 0.1 mM CaCl₂and loaded onto the HAP column at a flow rate of 100 cm/hr. The columnwas washed with 5 mM potassium phosphate pH 7.0, 100 mM NaCl, 0.1 mMCaCl₂, then 10 mM potassium phosphate pH 7.0, 100 mM NaCl, 0.1 mM CaCl₂pH. The protein was eluted with 70 mM potassium phosphate pH 7.0 andfiltered through a 0.22 μm filter into a 5 L sterile storage bag. Theeluate was tested for bioburden, protein concentration and enzymeactivity. The HAP-purified protein was then pumped through a 20 nM viralremoval filter via a pressure tank. The protein was added to the DV20pressure tank and filter (Pall Corporation), passing through an UltiporD V20 Filter with 20 nm pores (Pall Corporation) into a sterile 20 Lstorage bag. The filtrate was tested for protein concentration, enzymeactivity, oligosaccharide, monosaccharide and sialic acid profiling, andprocess-related impurities. The protein in the filtrate was thenconcentrated to 1 mg/mL using a 10 kD molecular weight cut off (MWCO)Sartocon Slice tangential flow filtration (TFF) system (Sartorius). Thefilter was first prepared by washing with a HEPES/saline solution (10 mMHEPES, 130 mM NaCl, pH 7.0) and the permeate was sampled for pH andconductivity. Following concentration, the concentrated protein wassampled and tested for protein concentration and enzyme activity. A 6×buffer exchange was performed on the concentrated protein into the finalbuffer: 10 mM HEPES, 130 mM NaCl, pH 7.0. The concentrated protein waspassed though a 0.22 μm filter into a 20 L sterile storage bag. Theprotein was sampled and tested for protein concentration, enzymeactivity, free sulfhydryl groups, oligosaccharide profiling andosmolarity.

Tables 23 to 29 provide monitoring data related to each of thepurification steps described above, for each 3D35M cell lot.

TABLE 23 Q sepharose column data Parameter HUA0406C HUA0410C HUA0415CHUA0420C Load volume 10647 13524 12852 20418 (mL) Load Volume/ 3.1 4.94.5 7.3 Resin Volume ratio Column 2770 3840 2850 2880 Volume (mL) Eluatevolume 6108 5923 5759 6284 (mL) Protein Conc. 2.8 3.05 2.80 2.86 ofEluate (mg/mL) Eluate 24493 26683 18321 21052 Enzyme Assay (U/mL) EnzymeYield 65 107 87 76 (%)

TABLE 24 Phenyl Sepharose column data Parameter HUA0406C HUA0410CHUA0415C HUA0420C Volume Before 5670 5015 5694 6251 Stock SolutionAddition (mL) Load Volume 7599 6693 7631 8360 (mL) Column 9106 9420 93409420 Volume (mL) Load Volume/ 0.8 0.71 0.82 0.89 Resin Volume ratioEluate volume 16144 18010 16960 17328 (mL) Protein Cone 0.4 0.33 0.330.38 of Eluate (mg/mL) Eluate Enzyme 8806 6585 4472 7509 Assay (U/mL)Protein Yield 41 40 36 37 (%) Enzyme Yield 102 88 82 96 (%)

TABLE 25 Amino Phenyl Boronate column data Parameter HUA0406C HUA0410CHUA0415C HUA0420C Load Volume 16136 17958 16931 17884 (mL) Load Volume/2.99 3.15 3.08 2.98 Resin Volume ratio Column 5400 5700 5500 5300 Volume(mL) Eluate volume 17595 22084 20686 19145 (mL) Protein Conc. 0.0 0.030.03 0.04 of Eluate (mg/mL) Protein Conc. not tested 0.03 0.00 0.04 ofFiltered Eluate (mg/mL) Eluate Enzyme 4050 2410 1523 4721 Assay (U/mL)Protein Yield 0 11 11 12 (%) Enzyme Yield not 41 40 69 (%) determined

TABLE 26 Hydroxyapatite column data Parameter HUA0406C HUA0410C HUA0415CHUA0420C Volume Before 16345 20799 20640 19103 Stock Solution Addition(mL) Load Volume/ 10.95 13.58 14.19 12.81 Resin Volume ratio Column 15001540 1462 1500 Volume (mL) Load volume 16429 20917 20746 19213 (mL)Eluate volume 4100 2415 1936 2419 (mL) Protein Conc. not tested 0.240.17 0.23 of Eluate (mg/mL) Protein Conc. NA NA 0.17 NA of FilteredEluate (mg/mL) Eluate Enzyme 14051 29089 20424 29826 Assay (U/mL)Protein Yield Not tested 93 53 73 (%) Enzyme Yield 87 118 140 104 (%)

TABLE 27 DV20 filtration data Parameter HUA0406C HUA0410C HUA0415CHUA0420C Start volume 4077 2233 1917 2419 (mL) Filtrate 4602 3334 29633504 Volume (mL) Protein Conc. 0.1 NA 0.09 NA of Filtrate (mg/mL)Protein Conc. NA 0.15 0.09 0.16 of Filtered Eluate (mg/mL) Protein Yieldnot tested 93 82 101 (%)

TABLE 28 Final concentration data Parameter HUA0406C HUA0410C HUA0415CHUA0420C Start volume 4575 3298 2963 3492 (mL) Concentrate 562 407 237316 Volume (mL) Protein Conc. 0.9 1.24 1.16 1.73 of Concentrate (mg/mL)Protein Yield 111 102 103 98 (%)

TABLE 29 Buffer Exchange into Final Formulation data Parameter HUA0406CHUA0410C HUA0415C HUA0420C Start Volume 562 407 237 316 (mL) FinalVolume 594 516 310 554 Buffer Exchanged Concentrate (mL) Protein Conc.1.00 0.97 0.98 1.00 of Concentrate (mg/mL) Protein Conc. 0.95 0.92 0.951.02 of Filtered Concentrate (mg/mL) Protein Yield 118 99 110 101 (%)

The purified and concentrated soluble rHuPH20 protein was asepticallyfilled into sterile vials with 5 mL and 1 mL fill volumes. The proteinwas passed though a 0.22 μm filter to an operator controlled pump thatwas used to fill the vials using a gravimetric readout. The vials wereclosed with stoppers and secured with crimped caps. The closed vialswere visually inspected for foreign particles and then labeled.Following labeling, the vials were flash-frozen by submersion in liquidnitrogen for no longer than 1 minute and stored at ≦−15° C. (−20±5° C.).

Example 5 Production Gen2 Cells Containing Soluble Human PH20 (rHuPH20)

The Gen1 3D35M cell line described in Example 2 was adapted to highermethotrexate levels to produce generation 2 (Gen2) clones. 3D35M cellswere seeded from established methotrexate-containing cultures into CDCHO medium containing 4 mM GlutaMAX-1™ and 1.0 μM methotrexate. Thecells were adapted to a higher methotrexate level by growing andpassaging them 9 times over a period of 46 days in a 37° C., 7% CO₂humidified incubator. The amplified population of cells was cloned outby limiting dilution in 96-well tissue culture plates containing mediumwith 2.0 μM methotrexate. After approximately 4 weeks, clones wereidentified and clone 3E10B was selected for expansion. 3E10B cells weregrown in CD CHO medium containing 4 mM GlutaMAX-1™ and 2.0 μMmethotrexate for 20 passages. A master cell bank (MCB) of the 3E10B cellline was created and frozen and used for subsequent studies.

Amplification of the cell line continued by culturing 3E10B cells in CDCHO medium containing 4 mM GlutaMAX-1™ and 4.0 μM methotrexate. Afterthe 12^(th) passage, cells were frozen in vials as a research cell bank(RCB). One vial of the RCB was thawed and cultured in medium containing8.0 μM methotrexate. After 5 days, the methotrexate concentration in themedium was increased to 16.0 μM, then 20.0 μM 18 days later. Cells fromthe 8^(th) passage in medium containing 20.0 μM methotrexate were clonedout by limiting dilution in 96-well tissue culture plates containing CDCHO medium containing 4 mM GlutaMAX-1™ and 20.0 μM methotrexate. Cloneswere identified 5-6 weeks later and clone 2B2 was selected for expansionin medium containing 20.0 μM methotrexate. After the 11th passage, 2B2cells were frozen in vials as a research cell bank (RCB).

The resultant 2B2 cells are dihydrofolate reductase deficient (dhfr-)DG44 CHO cells that express soluble recombinant human PH20 (rHuPH20).The soluble PH20 is present in 2B2 cells at a copy number ofapproximately 206 copies/cell. Southern blot analysis of Spe I-, Xba I-and BamH I/Hind III-digested genomic 2B2 cell DNA using arHuPH20-specific probe revealed the following restriction digestprofile: one major hybridizing band of ˜7.7 kb and four minorhybridizing bands (˜13.9, ˜6.6, ˜5.7 and ˜4.6 kb) with DNA digested withSpe I; one major hybridizing band of ˜5.0 kb and two minor hybridizingbands (˜13.9 and ˜6.5 kb) with DNA digested with Xba I; and one singlehybridizing band of ˜1.4 kb observed using 2B2 DNA digested with BamHI/Hind III. Sequence analysis of the mRNA transcript indicated that thederived cDNA (SEQ ID NO:56) was identical to the reference sequence (SEQID NO:49) except for one base pair difference at position 1131, whichwas observed to be a thymidine (T) instead of the expected cytosine (C).This is a silent mutation, with no effect on the amino acid sequence.

Example 6 A. Production of Gen2 Soluble rHuPH20 in 300 L Bioreactor CellCulture

A vial of HZ24-2B2 was thawed and expanded from shaker flasks through 36L spinner flasks in CD-CHO media (Invitrogen, Carlsbad, Calif.)supplemented with 20 μM methotrexate and GlutaMAX-1™ (Invitrogen).Briefly, the vial of cells was thawed in a 37° C. water bath, media wasadded and the cells were centrifuged. The cells were re-suspended in a125 mL shake flask with 20 mL of fresh media and placed in a 37° C., 7%CO₂ incubator. The cells were expanded up to 40 mL in the 125 mL shakeflask. When the cell density reached greater than 1.5×10⁶ cells/mL, theculture was expanded into a 125 mL spinner flask in a 100 mL culturevolume. The flask was incubated at 37° C., 7% CO₂. When the cell densityreached greater than 1.5×10⁶ cells/mL, the culture was expanded into a250 mL spinner flask in 200 mL culture volume, and the flask wasincubated at 37° C., 7% CO₂. When the cell density reached greater than1.5×10⁶ cells/mL, the culture was expanded into a 1 L spinner flask in800 mL culture volume and incubated at 37° C., 7% CO₂. When the celldensity reached greater than 1.5×10⁶ cells/mL the culture was expandedinto a 6 L spinner flask in 5000 mL culture volume and incubated at 37°C., 7% CO₂. When the cell density reached greater than 1.5×106 cells/mLthe culture was expanded into a 36 L spinner flask in 32 L culturevolume and incubated at 37° C., 7% CO₂.

A 400 L reactor was sterilized and 230 mL of CD-CHO media was added.Before use, the reactor was checked for contamination. Approximately 30L cells were transferred from the 36 L spinner flasks to the 400 Lbioreactor (Braun) at an inoculation density of 4.0×10⁵ viable cells perml and a total volume of 260 L. Parameters were temperature setpoint,37° C.; Impeller Speed 40-55 RPM; Vessel Pressure: 3 psi; Air Sparge0.5-1.5 L/Min.; Air Overlay: 3 L/min. The reactor was sampled daily forcell counts, pH verification, media analysis, protein production andretention. Also, during the run nutrient feeds were added. At 120 hrs(day 5), 10.4 L of Feed #1 Medium (4×CD-CHO+33 g/L Glucose+160 mL/LGlutamax-1™+83 mL/L Yeastolate+33 mg/L rHuInsulin) was added. At 168hours (day 7), 10.8 L of Feed #2 (2×CD-CHO+33 g/L Glucose+80 mL/LGlutamax-1™+167 mL/L Yeastolate+0.92 g/L Sodium Butyrate) was added, andculture temperature was changed to 36.5° C. At 216 hours (day 9), 10.8 Lof Feed #3 (1×CD-CHO+50 g/L Glucose+50 mL/L Glutamax-1™+250 mL/LYeastolate+1.80 g/L Sodium Butyrate) was added, and culture temperaturewas changed to 36° C. At 264 hours (day 11), 10.8 L of Feed #4(1×CD-CHO+33 g/L Glucose+33 mL/L Glutamax-1™+250 mL/L Yeastolate+0.92g/L Sodium Butyrate) was added, and culture temperature was changed to35.5° C. The addition of the feed media was observed to dramaticallyenhance the production of soluble rHuPH20 in the final stages ofproduction. The reactor was harvested at 14 or 15 days or when theviability of the cells dropped below 40%. The process resulted in afinal productivity of 17,000 Units per ml with a maximal cell density of12 million cells/mL. At harvest, the culture was sampled for mycoplasma,bioburden, endotoxin and viral in vitro and in vivo, TransmissionElectron Microscopy (TEM) and enzyme activity.

The culture was pumped by a peristaltic pump through four Millistakfiltration system modules (Millipore) in parallel, each containing alayer of diatomaceous earth graded to 4-8 μm and a layer of diatomaceousearth graded to 1.4-1.1 μm, followed by a cellulose membrane, thenthrough a second single Millistak filtration system (Millipore)containing a layer of diatomaceous earth graded to 0.4-0.11 μm and alayer of diatomaceous earth graded to <0.1 μm, followed by a cellulosemembrane, and then through a 0.22 μm final filter into a sterile singleuse flexible bag with a 350 L capacity. The harvested cell culture fluidwas supplemented with 10 mM EDTA and 10 mM Tris to a pH of 7.5. Theculture was concentrated 10× with a tangential flow filtration (TFF)apparatus using four Sartoslice TFF 30 kDa molecular weight cut-off(MWCO) polyether sulfone (PES) filter (Sartorious), followed by a 10×buffer exchange with 10 mM Tris, 20 mM Na₂SO₄, pH 7.5 into a 0.22 μmfinal filter into a 50 L sterile storage bag.

The concentrated, diafiltered harvest was inactivated for virus. Priorto viral inactivation, a solution of 10% Triton X-100, 3% tri (n-butyl)phosphate (TNBP) was prepared. The concentrated, diafiltered harvest wasexposed to 1% Triton X-100, 0.3% TNBP for 1 hour in a 36 L glassreaction vessel immediately prior to purification on the Q column.

B. Purification of Gen2 soluble rHuPH20

A Q Sepharose (Pharmacia) ion exchange column (9 L resin, H=29 cm, D=20cm) was prepared. Wash samples were collected for a determination of pH,conductivity and endotoxin (LAL) assay. The column was equilibrated with5 column volumes of 10 mM Tris, 20 mM Na2SO4, pH 7.5. Following viralinactivation, the concentrated, diafiltered harvest was loaded onto theQ column at a flow rate of 100 cm/hr. The column was washed with 5column volumes of 10 mM Tris, 20 mM Na2SO4, pH 7.5 and 10 mM HEPES, 50mM NaCl, pH 7.0. The protein was eluted with 10 mM HEPES, 400 mM NaCl,pH 7.0 into a 0.22 μm final filter into sterile bag. The eluate samplewas tested for bioburden, protein concentration and hyaluronidaseactivity. A₂₈₀ absorbance reading were taken at the beginning and end ofthe exchange.

Phenyl-Sepharose (Pharmacia) hydrophobic interaction chromatography wasnext performed. A Phenyl-Sepharose (PS) column (19-21 L resin, H=29 cm,D=30 cm) was prepared. The wash was collected and sampled for pH,conductivity and endotoxin (LAL assay). The column was equilibrated with5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate,0.1 mM CaCl₂, pH 7.0. The protein eluate from the Q sepharose column wassupplemented with 2M ammonium sulfate, 1 M potassium phosphate and 1 MCaCl₂ stock solutions to yield final concentrations of 5 mM, 0.5 M and0.1 mM, respectively. The protein was loaded onto the PS column at aflow rate of 100 cm/hr and the column flow thru collected. The columnwas washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1mM CaCl2 pH 7.0 at 100 cm/hr and the wash was added to the collectedflow thru. Combined with the column wash, the flow through was passedthrough a 0.22 μm final filter into a sterile bag. The flow through wassampled for bioburden, protein concentration and enzyme activity.

An aminophenyl boronate column (Prometics) was prepared. The wash wascollected and sampled for pH, conductivity and endotoxin (LAL assay).The column was equilibrated with 5 column volumes of 5 mM potassiumphosphate, 0.5 M ammonium sulfate. The PS flow through containingpurified protein was loaded onto the aminophenyl boronate column at aflow rate of 100 cm/hr. The column was washed with 5 mM potassiumphosphate, 0.5 M ammonium sulfate, pH 7.0. The column was washed with 20mM bicine, 0.5 M ammonium sulfate, pH 9.0. The column was washed with 20mM bicine, 100 mM sodium chloride, pH 9.0. The protein was eluted with50 mM HEPES, 100 mM NaCl, pH 6.9 and passed through a sterile filterinto a sterile bag. The eluted sample was tested for bioburden, proteinconcentration and enzyme activity.

The hydroxyapatite (HAP) column (Biorad) was prepared. The wash wascollected and tested for pH, conductivity and endotoxin (LAL assay). Thecolumn was equilibrated with 5 mM potassium phosphate, 100 mM NaCl, 0.1mM CaCl₂, pH 7.0. The aminophenyl boronate purified protein wassupplemented to final concentrations of 5 mM potassium phosphate and 0.1mM CaCl₂ and loaded onto the HAP column at a flow rate of 100 cm/hr. Thecolumn was washed with 5 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1mM CaCl₂. The column was next washed with 10 mM potassium phosphate, pH7, 100 mM NaCl, 0.1 in M CaCl₂. The protein was eluted with 70 mMpotassium phosphate, pH 7.0 and passed through a 0.22 μm sterile filterinto a sterile bag. The eluted sample was tested for bioburden, proteinconcentration and enzyme activity.

The HAP purified protein was then passed through a viral removal filter.The sterilized Virosart filter (Sartorius) was first prepared by washingwith 2 L of 70 mM potassium phosphate, pH 7.0. Before use, the filteredbuffer was sampled for pH and conductivity. The HAP purified protein waspumped via a peristaltic pump through the 20 nM viral removal filter.The filtered protein in 70 mM potassium phosphate, pH 7.0 was passedthrough a 0.22 μm final filter into a sterile bag. The viral filteredsample was tested for protein concentration, enzyme activity,oligosaccharide, monosaccharide and sialic acid profiling. The samplealso was tested for process related impurities.

The protein in the filtrate was then concentrated to 10 mg/mL using a 10kD molecular weight cut off (MWCO) Sartocon Slice tangential flowfiltration (TFF) system (Sartorius). The filter was first prepared bywashing with 10 mM histidine, 130 mM NaCl, pH 6.0 and the permeate wassampled for pH and conductivity. Following concentration, theconcentrated protein was sampled and tested for protein concentrationand enzyme activity. A 6× buffer exchange was performed on theconcentrated protein into the final buffer: 10 mM histidine, 130 mMNaCl, pH 6.0. Following buffer exchange, the concentrated protein waspassed though a 0.22 μm filter into a 20 L sterile storage bag. Theprotein was sampled and tested for protein concentration, enzymeactivity, free sulfhydryl groups, oligosaccharide profiling andosmolarity.

The sterile filtered bulk protein was then asceptically dispensed at 20mL into 30 mL sterile Teflon vials (Nalgene). The vials were then flashfrozen and stored at −20±5° C.

C. Comparison of Production and Purification of Gen1 Soluble rHuPH20 andGen2 Soluble rHuPH20

The production and purification of Gen2 soluble rHuPH20 in a 300 Lbioreactor cell culture contained some changes in the protocols comparedto the production and purification Gen1 soluble rHuPH20 in a 100 Lbioreactor cell culture (described in Example 4B). Table 30 sets forthexemplary differences, in addition to simple scale up changes, betweenthe methods.

TABLE 30 Process Difference Gen1 soluble rHuPH20 Gen2 soluble rHuPH20Cell line 3D35M 2B2 Media used to expand cell Contains 0.10 μM Contains20 μM inoculum methotrexate (0.045 mg/L) methotrexate (9 mg/L) Media in6 L cultures Contains 0.10 μM Contains no methotrexate onwardsmethotrexate 36 L spinner flask No instrumentation Equipped with 20 Loperating volume. instrumentation that monitors and controls pH,dissolved oxygen, sparge and overlay gas flow rate. 32 L operatingvolume Final operating volume in Approx. 100 L in a 125 L Approx. 300 Lin a 400 L bioreactor bioreactor bioreactor (initial culture (initialculture volume + volume + 260 L) 65 L) Culture media in final NorHuInsulin 5.0 mg/L rHuInsulin bioreactor Media feed volume Scaled at 4%of the Scaled at 4% of the bioreactor cell culture bioreactor cellculture volume i.e. 3.4, 3.5 and 3.7 L, volume i.e. 10.4, 10.8,resulting in a target 11.2 and 11.7 L, resulting bioreactor volume of~92 L. in a target bioreactor volume of ~303 L. Media feed Feed #1Medium: CD Feed #1 Medium: 4x CD CHO + 50 g/L Glucose + CHO + 33 g/LGlucose + 8 mM GlutaMAX ™-1 32 mM Glutamax + 16.6 g/L Feed #2 (CD CHO +50 g/L Yeastolate + 33 mg/L Glucose + 8 mM rHuInsulin GlutaMAX + 1.1 g/LFeed #2: 2x CD CHO + 33 g/L Sodium Butyrate Glucose + 16 mM Feed #3: CDCHO + 50 g/L Glutamax + 33.4 g/L Glucose + 8 mM Yeastolate + 0.92 g/LGlutaMAX + 1.1 g/L Sodium Butyrate Sodium Butyrate Feed #3: 1x CD CHO +50 g/L Glucose + 10 mM Glutamax + 50 g/L Yeastolate + 1.80 g/L SodiumButyrate Feed #4: 1x CD CHO + 33 g/L Glucose + 6.6 mM Glutamax + 50 g/LYeastolate + 0.92 g/L Sodium Butyrate Filtration of bioreactor cell Fourpolyethersulfone 1^(st) stage - Four modules in culture filters (8.0 μm,0.65 μm, parallel, each with a layer 0.22 μm and 0.22 μm) in ofdiatomaceous earth series graded to 4-8 μm and a 100 L storage bag layerof diatomaceous earth graded to 1.4-1.1 μm, followed by a cellulosemembrane. 2^(nd) stage - single module containing a layer ofdiatomaceous earth graded to 0.4-0.11 μm and a layer of diatomaceousearth graded to <0.1 μm, followed by a cellulose membrane. 3^(rd)stage - 0.22 μm polyethersulfone filter 300 L storage bag Harvested cellculture is supplemented with 10 mM EDTA, 10 mM Tris to a pH of 7.5.Concentration and buffer Concentrate with 2 TFF Concentrate using fourexchange prior to with Millipore Spiral Sartorius Sartoslice TFFchromatography Polyethersulfone 30K 30K MWCO Filter MWCO Filter BufferExchange the Buffer Exchange the Concentrate 10× with 10 mM Concentrate6× with 10 mM Tris, 20 mM Na2SO4, HEPES, 25 mM pH 7.5 NaCl, pH 7.0 50 Lsterile storage bag 20 L sterile storage bag Viral inactivation prior toNone Viral inactivation chromatography performed with the addition of a1% Triton X- 100, 0.3% Tributyl Phosphate, pH 7.5, 1^(st) purificationstep (Q No absorbance reading A280 measurements at the sepharose)beginning and end Viral filtration after Pall DV-20 filter (20 nm)Sartorius Virosart filter (20 nm) chromatography Concentration andbuffer HEPES/saline pH 7.0 Histidine/saline, pH 6.0 exchange afterbuffer buffer chromatography Protein concentrated to 1 mg/ml Proteinconcentrated to 10 mg/ml

Example 7 Determination of Sialic Acid and Monosaccharide Content

The sialic acid and monosaccharide content of soluble rHuPH20 can beassessed by reverse phase liquid chromatography (RPLC) followinghydrolysis with trifluoroacetic acid. In one example, the sialic acidand monosaccharide content of purified hyaluronidase lot # HUB0701E (1.2mg/mL; produced and purified essentially as described in Example 6) wasdetermined. Briefly, 100 μg sample was hydrolyzed with 40% (v/v)trifluoroacetic acid at 100° C. for 4 hours in duplicate. Followinghydrolysis, the samples were dried down and resuspended in 300 μL water.A 45 μL aliquot from each re-suspended sample was transferred to a newtube and dried down, and 10 μL of a 10 mg/mL sodium acetate solution wasadded to each. The released monosaccharides were fluorescently labeledby the addition of 50 μL of a solution containing 30 mg/mL2-aminobenzoic acid, 20 mg/mL sodium cyanoborohydride, approximately 40mg/mL sodium acetate and 20 mg/mL boric acid in methanol. The mixturewas incubated for 30 minutes at 80° C. in the dark. The derivitizationreaction was quenched by the addition of 440 μL of mobile phase A (0.2%(v/v) n-butylamine, 0.5% (v/v) phosphoric acid, 1% (v/v)tetrahydrofuran). A matrix blank of water also was hydrolyzed andderivitized as described for the hyaluronidase sample as a negativecontrol. The released monosaccharides were separated by RPLC using anOctadecyl (C₁₈) reverse phase column (4.6×250 mm, 5 μm particle size; J.T. Baker) and monitored by fluorescence detection (360 nm excitation,425 nm emission). Quantitation of the monosaccharide content was made bycomparison of the chromatograms from the hyaluronidase sample withchromatograms of monosaccharide standards including N-D-glucosamine(GlcN), N-D-galactosamine (GalN), galactose, fucose and mannose. Table31 presents the molar ratio of each monosaccharide per hyaluronidasemolecule.

TABLE 31 Monosaccharide content of soluble rHuPH20 Lot Replicate GlcNGalN Galactose Mannose Fucose HUB0701E 1 14.28 0.07* 6.19 25.28 2.69 213.66 0.08* 6.00 24.34 2.61 Average 13.97 0.08* 6.10 24.81 2.65 *GalNresults were below the limit of detection

Example 8 C-Terminal Heterogeneity of Soluble rHuPH20 from 3D35M and 2B2Cells

C-terminal sequencing was performed on two lots of sHuPH20 produced andpurified from 3D35M cells in a 100 L bioreactor volume (Lot HUA0505MA)and 2B2 cells in a 300 L bioreactor volume (Lot HUB0701EB). The lotswere separately digested with endoproteinase Asp-N, which specificallycleaves peptide bonds N-terminally at aspartic and cysteic acid. Thisreleases the C-terminal portion of the soluble rHuPH20 at the asparticacid at position 431 of SEQ ID NO:4. The C-terminal fragments wereseparated and characterized to determine the sequence and abundance ofeach population in Lot HUA0505MA and Lot HUB0701EB.

It was observed that the soluble rHuPH20 preparations from 3D35M cellsand 2B2 cells displayed heterogeneity, and contained polypeptides thatdiffered from one another in their C-terminal sequence (Tables 27 and28). This heterogeneity is likely the result of C-terminal cleavage ofthe expressed 447 amino acid polypeptide (SEQ ID NO:4) by peptidasespresent in the cell culture medium or other solutions during theproduction and purification process. The polypeptides in the solublerHuPH20 preparations have amino acid sequences corresponding to aminoacids 1-447, 1-446, 1-445, 1-444 and 1-443 of the soluble rHuPH20sequence set forth SEQ ID NO:4. The full amino acid sequence of each ofthese polypeptides is forth in SEQ ID NOS: 4 to 8, respectively. Asnoted in tables 32 and 33, the abundance of each polypeptide in thesoluble rHuPH20 preparations from 3D35M cells and 2B2 cells differs.

TABLE 32 Analysis of C-terminal fragments from Lot HUA0505MA Amino acid  position (relative Frag- to SEQ Theor. Exp. Elution Abun- mentID NO: 4) Sequence mass Mass Error time dance D28a 431-447DAFKLPPMETEEPQIFY 2053.97 2054.42 0.45 99.87  0.2% (SEQ ID NO: 57) D28b431-446 DAFKLPPMETEEPQIF 1890.91 1891.28 0.37 97.02 18.4%(SEQ ID NO: 58) D28c 431-445 DAFKLPPMETEEPQI 1743.84 1744.17 0.33 86.4 11.8% (SEQ ID NO: 59) D28d 431-444 DAFKLPPMETEEPQ 1630.70 1631.07 0.3274.15 56.1% (SEQ ID NO: 60) D28e 431-443 DAFKLPPMETEEP 1502.70 1502.980.28 77.36 13.6% (SEQ ID NO: 61) D28f 431-442 DAFKLPPMETEE 1405.64 NDN/A N/A  0.0% (SEQ ID NO: 62)

TABLE 33 Analysis of C-terminal fragments from Lot HUB0701E13 Amino acidposition (relative Frag- to SEQ Theor. Exp. Elution Abun- ment ID NO: 4)Sequence mass Mass Error time dance D28a 431-477 DAFKLPPMETEEPQIFY2053.97 2054.42 0.45 99.89  1.9% (SEQ ID NO: 57) D28b 431-446DAFKLPPMETEEPQIF 1890.91 1891.36 0.45 96.92 46.7% (SEQ ID NO: 58) D28c431-445 DAFKLPPMETEEPQI 1743.84 1744.24 0.40 85.98 16.7% (SEQ ID NO: 59)D28d 431-444 DAFKLPPMETEEPQ 1630.70 1631.14 0.39 73.9  27.8%(SEQ ID NO: 60) D28e 431-443 DAFKLPPMETEEP 1502.70 1503.03 0.33 77.02 6.9% (SEQ ID NO: 61) D28f 431-442 DAFKLPPMETEE 1405.64 ND N/A N/A  0.0%(SEQ ID NO: 62)

Example 9 Comparison of the Dispersion Activity of Different HyaluronanDegrading Enzymes

The ability of different hyaluronan degrading enzymes to act as adispersion agent was assessed in vivo. A dispersion assay in mice wasused to assess the ability of different hyaluronan degrading enzyme toact as dispersion agents of trypan blue, and also to assess the abilityof the enzymes to enhance the efficacy of co-administered insulin inreducing blood glucose levels. The hyaluronan degrading enzymes assayedincluded rHuPH20, pegylated PH20 (PEG PH20), Hyal 1, Chondroitinase ABC,Chondroitinase AC and Streptomyces hyalurolyticus lyase. These weremixed with trypan blue and Humulin® insulin in a neutral buffer (10 mMsodium phosphate, pH 7.4, 145.5 mM NaCl, 1 mg/ml human serum albumin)and delivered to anesthetized mice. Both the area of dispersion of thetrypan blue and the blood glucose levels were then measured. The neutralpH buffer alone and Humulin® insulin alone were used a negativecontrols. The ability of a low pH buffer (pH 4.5) to act as a dispersionagent also was examined.

Nine groups of NCr nu/nu homozygous mice, approximately 10 weeks of ageand with body weights of 21-25 g, with 3 mice per group, wereanesthetized by intraperitoneal injection of ketamine/xylazine (10:1mixture in saline). Thereafter, the mice were administered 40 μL of ahyaluronan degrading enzyme and 5 Units/mL Humulin® insulin with 0.4%Trypan Blue dye by intradermal injection at the midline over the caudalend of the ribcage. Control groups administered Humulin® insulin alone,buffer alone or buffer and Humulin® insulin also were included.Specifically, group 1 mice were the negative control and received trypanblue with a neutral pH buffer; group 2 mice received trypan blue with 5Units/mL Humulin® insulin in a low pH buffer; group 3 mice receivedtrypan blue with 5 Units/mL Humulin® insulin and 10 Units/mL rHuPH20;group 4 mice received trypan blue with 5 Units/mL Humulin® insulin and10 Units/mL PEG PH20 (generated as described in Example 10, below);group 5 mice received trypan blue with 5 Units/mL Humulin® insulin and10 Units/mL Hyal 1; group 6 mice received trypan blue with 5 Units/mLHumulin® insulin and 10 Units/mL Chondroitinase ABC (Associates of CapeCod, E. Falmouth, Mass.); group 7 mice received trypan blue with 5Units/mL Humulin® insulin and 1 Unit/mL Condroitinase AC (Associates ofCape Cod, E. Falmouth, Mass.); group 8 mice received trypan blue with 5Units/mL Humulin® insulin and 100 Units/mL Streptomyces hyalurolyticuslyase (Calbiochem); group 9 mice received trypan blue with 5 Units/mLHumulin® insulin. The dispersion of the trypan blue dye was thenmeasured by a caliper at 2.5, 5, 10, 15 and 20 minutes post injection.The dye dispersion area (mm²) was calculated by multiplying the longestaxis M1 (length of the dye front) and M2 (width of the dye front) by ¼π(M1M2×¼π). The blood glucose levels were measured using a glucometer at0, 5, 10, 15 and 20 minutes

1. Dye Dispersion

Table 34 sets forth the mean dye dispersion area followingadministration of each of the test articles. The trypan blue dye inneutral pH buffer and low pH buffer exhibit minimal spreading, with thedispersion area ranging from an average of about 36 mm² at 2.5 minutespost injection to about 51 mm² at 20 minutes post injection. When thetrypan blue dye was mixed and delivered with Humulin® insulin, Hyal 1,or PEG PH20, there was no statistically significant increase in thedispersion area compared to that observed when the dye was mixed withbuffer only. In contrast, a significant increase in the dispersion ofthe dye was observed when mixed and delivered with rHuPH20,Chondroitinase ABC, Chondroitinase AC or Streptomyces hyalurolyticuslyase. The average dispersion area of trypan blue dye when mixed anddelivered with rHuPH20 was about 45 mm², 66 mm², 80 mm², 86 mm² and 102mm² at 2.5, 5, 10, 15 and 20 minutes after injection, respectively. Theaverage dispersion area of trypan blue dye when mixed and delivered withCondroitinase AC was about 76 mm², 107 mm², 107 mm², 110 mm² and 116 mm²at 2.5, 5, 10, 15 and 20 minutes after injection, respectively. Theaverage dispersion area of trypan blue dye when mixed and delivered withCondroitinase ABC was about 57 mm², 75 mm², 79 mm², 81 mm² and 88 mm² at2.5, 5, 10, 15 and 20 minutes after injection, respectively. The averagedispersion area of trypan blue dye when mixed and delivered withStreptomyces hyalurolyticus lyase was about 74 mm², 76 mm², 101 mm², 103mm² and 130 mm² at 2.5, 5, 10, 15 and 20 minutes after injection,respectively.

TABLE 34 Group Mean Summary of Dye Dispersion Areas (mm²) Dye DispersionAreas (mm²) 2.5 5 10 15 20 Group Test Article min min min min min 1Neutral pH 36.11 41.63 47.19 52.47 51.34 Buffer Vehicle/ Control 2 LowpH 33.34 34.47 41.88 44.91 51.18 Buffer 3 PEG PH20 37.28 47.08 52.4054.94 58.17 (10 U/mL) 4 rHuPH20 44.58 66.02 79.46 86.24 101.90 (10 U/mL)5 Hyal I 31.53 36.27 39.60 46.41 48.21 (10 U/mL) 6 Chondroitinase 56.8575.06 78.60 81.44 87.85 ABC (10 U/mL) 7 Chondroitinase 75.67 106.56106.49 110.43 115.82 AC (1 U/mL) 8 Strep lyase 73.97 75.58 101.03 102.69129.56 (100 U/mL) 9 Humulin R 38.22 43.94 50.76 52.49 58.41

2. Blood Glucose Levels

Table 35 sets forth the mean blood glucose levels (mg/dL) followingadministration of each test article. The blood glucose levels in miceadministered dye and buffer only increased from an average ofapproximately 212 mg/dL prior to injection to approximately 332 mg/dL at5 minutes post injection. Thereafter, the levels gradually rose toapproximately 367 mg/dL at 20 minutes post injection. This increase ofblood glucose in the absence of insulin is due to a well known effect ofanesthetics on blood glucose in rodents (see, e.g. Saha et al., (2005)Exp. Biol. Med. 230:777-784). When Humulin® insulin was administered,the blood glucose levels rose briefly to an average of about 292 mg/dLat 5 minutes post injection (from average of about 226 mg/dL prior toinjection) before dropping to an average of about 171 mg/dL, 122 mg/dLand 97 mg/dL at 10, 15 and 20 minutes post injection, respectively.While all of the hyaluronan degrading enzymes lowered blood glucoselevels when administered with Humulin® insulin, co-administration ofrHuPH20, PEG PH20, Chondroitinase ABC and Streptomyces hyalurolyticuslyase appeared to reduce levels even faster than observed with Humulin®insulin alone,

TABLE 35 Group Mean Summary of Blood Glucose Level (mg/dL) Blood GlucoseLevel (mg/dL) 0 5 10 15 20 Group Test Article min min min min min 1Neutral pH 212.00 332.00 344.00 3.61.33 367.67 Buffer Vehicle/ Control 2Low pH 196.67 259.67 249.33 231.33 220.67 Buffer 3 PEG PH20 170.00196.67 110.00 68.33 46.67 (10 U/mL) 4 rHuPH20 165.67 173.00 96.00 63.6739.00 (10 U/mL) 5 Hyal I 155.67 201.33 144.33 77.00 52.67 (10 U/mL) 6Chondroitinase 129.67 123.67 65.00 44.67 21.00 ABC (10 U/mL) 7Chondroitinase 174.33 248.67 204.67 165.33 133.67 AC (1 U/mL) 8 Streplyase 140.33 120.67 68.67 41.00 27.67 (100 U/mL) 9 Humulin R 226.33292.00 171.33 122.33 96.67

Example 10 PEGylation of rHuPH20

A. Conjugation of mPEG-SBA-30K to rHuPH20

In order to generate a PEGylated soluble human hyaluronidase, rHuPH20(which is approximately 60 KDa in size) was covalently conjugated to alinear N-hydroxysuccinimidyl ester of methoxy poly(ethylene glycol)butanoic acid (mPEG-SBA-30K), having an approximate molecular weight of30 kDa. The structure of mPEG-SBA is shown in scheme 2, below:

Methods used to prepare the mPEG-SBA-30K that was used to PEGylaterHuPH20 are described, for example, in U.S. Pat. No. 5,672,662).Briefly, the mPEG-SBA-30K is made according to the following procedure:

A solution of ethyl malonate (2 equivalents) dissolved in dioxane isadded drop by drop to sodium hydride (2 equivalents) and toluene under anitrogen atmosphere. mPEG methane sulfonate (1 equivalent, MW 30 kDa,Shearwater) is dissolved in toluene and added to the above mixture. Theresulting mixture is refluxed for approximately 18 hours. The reactionmixture is concentrated to half its original volume, extracted with 10%aqueous NaCl solution, extracted with 1% aqueous hydrochloric acid, andthe aqueous extracts are combined. The collected aqueous layers areextracted with dichloromethane (3×) and the organic layer is dried withmagnesium sulfate, filtered and evaporated to dryness. The resultingresidue is dissolved in 1N sodium hydroxide containing sodium chlorideand the mixture is stirred for 1 hour. The pH of the mixture is adjustedto approximately 3 by addition of 6N hydrochloric acid. The mixture isextracted with dichloromethane (2×).

The organic layer is dried over magnesium sulfate, filtered,concentrated, and poured into cold diethyl ether. The precipitate iscollected by filtration and dried under vacuum. The resulting compoundis dissolved in dioxane and refluxed for 8 hours and then concentratedto dryness. The resulting residue is dissolved in water and extractedwith dichloromethane (2×), dried over magnesium sulfate, and thesolution is concentrated by rotary evaporation and then poured into colddiethyl ether. The precipitate is collected by filtration and driedunder vacuum. The resulting compound (1 equivalent) is dissolved indichloromethane and N-hydroxysuccinimide (2.1 equivalents) is added. Thesolution is cooled to 0° C. and a solution of dicyclohexylcarbodiimide(2.1 equivalents) in dichloromethane is added dropwise. The solution isstirred at room temperature for approximately 18 hours. The reactionmixture is filtered, concentrated and precipitated in diethyl ether. Theprecipitate is collected by filtration and dried under vacuum to affordmPEG-SBA-30K.

To make the PEGylated rHuPH20, mPEG-SBA-30K was coupled to the aminogroup(s) of rHuPH20 by covalent conjugation, providing stable amidebonds between rHuPH20 and mPEG, as shown in Scheme 3.

For the conjugation, the mPEG-SBA-30K was added in powder form torHuPH20 (at a concentration of 10 mg/mL in 130 mM NaCl/10 mM HEPES; pH7). The PEG:rHuPH20 ratio was 10:1 (molar ratio). After the PEG haddissolved in the buffer, the solution was sterile-filtered (Corning 50mL Tube top filter, polystyrene, cellulose acetate 0.22 μm membrane).The conjugation was carried out overnight, with stirring, at 4° C. in acold room.

Following conjugation, the solution was concentrated, using a 100,000MWCO TFF membrane, and buffer exchanged against 130 mM NaCl/10 mM HEPESat pH 6.8. The resulting material, which was tested for enzyme activity,as described in Example 2, above, was diluted using 130 mM NaCl/10 mMHEPES at pH 6.8 to obtain a final enzyme activity of 100,000 U/mL(corresponding to approximately 2.5 mg peptide/mL). This PEGylatedrHuPH20 material was filled, in 1 mL volumes, into a 13-mm Type-1 glassvial with brombutyl seal, and stored frozen (frozen overnight in a −80°C. freezer, then put in a −20° C. freezer for longer storage).

B. Analysis of PEGylated rHuPH20

The PEGylated rHuPH20 material was assayed by gel electrophoresis. Threebatches of PEGylated rHuPH20, made as in Example 7A above, revealed anidentical pattern of multiple bands, representing unreacted PEG andmultiple species of mPEG-rHuPH20 conjugates, which migrated at differentdistances. Based on comparison with migration of a molecular weightmarker, the bands representing the species ranged from approximately 90KDa to 300 KDa, with three dark bands migrating above the 240 KDamarker. These data indicated that the PEGylated rHuPH20, generated bycovalent conjugation of mPEG-SBA-30K, contained a heterogeneous mixtureof PEGylated rHuPH20 species, likely including mono-, di- andtri-PEGylated proteins. The lack of a visible band at 60 KDa suggestedthat all the protein had reacted with the PEG, and that no detectablenative rHuPH20 was present in the mixture.

Example 11 Effect of rHuPH20 on the Pharmacokinetics of InsulinFollowing Subcutaneous Administration in Pigs

To determine whether a pig model would be suitable for modeling thepharmacokinetics of prandial insulins coadministered with recombinanthyaluronidase (e.g. rHuPH20), the pharmacokinetics of Humalog® insulinlispro and Humulin® R insulin after subcutaneous injection with orwithout rHuPH20 in pigs was assessed. The results were then compared tothose observed in humans (see Example 1), to determine whether the pigmodel accurately reflected that seen in humans.

Briefly, Humalog® insulin lispro and Humulin® R insulin, with andwithout rHuPH20, were administered subcutaneously to six pigs in arandomized, 4-way crossover study. Each animal received three cycles oftreatment with all four test articles to facilitate comparison of thereproducibility of the insulin pharmacokinetics over a series of dosingcycles. Blood samples were collected and the serum was assessed for todetermine the levels of immunoreactive insulin (IRI). Variouspharmacokinetic parameters, including t_(max), C_(max), Early t_(50%),Late t_(50%), and AUC_(max) were then determined.

A. Dosing and Sampling

Dosing solutions (or test articles) of 100 U/mL Humalog® insulin lisproor Humulin® R insulin, with and without 4800 U/mL rHuPH20, were preparedas follows. The 100 Humalog® insulin lispro alone and Humulin® R insulinalone solutions were prepared from commercial lots of Humalog® insulinlispro (100 U/mL; Lot A418976, Eli Lilly) and Humulin® R insulin (100U/mL; Lot A393318, Eli Lilly, diluted 1:5 with Sterile Diluent (EliLilly), respectively. To prepare the Humalog® insulin lispro/rHuPH20solution, 910 μL of 100 U/mL Humalog® insulin lispro (Eli Lilly, LotA418976), 44.6 mL HYLENEX recombinant (hyaluronidase human injection)(Baxter, Lot 903646) and 45.4 μL rHuPH20 API 1 mg/mL (HalozymeTherapeutics, Lot HUA0703MA) was mixed for a final Humalog® insulinlispro concentration of 91 U/mL and a hyaluronidase activity of 5454U/mL. To prepare the Humulin® R insulin/rHuPH20 solution, 200 tit of 500U/mL Humulin® R insulin (Eli Lilly, Lot A393318) and 800 μL rHuPH20 DrugProduct 6000 U/mL (Halozyme, Lot 288004; rHuPH20 Drug Product contained50 μg rHuPH20 in 145 mM NaCl, 10 mM Sodium Phosphate Dibasic, 2.7 mMCalcium Chloride, 2.7 mM EDTA Disodium Salt, 1 mg/mL Human SerumAlbumin, pH 7.4) was mixed for a final insulin concentration of 100 U/mLand rHuPH20 hyaluronidase activity of 4800 U/mL.

The solutions containing rHuPH20 were sterile filtered and filled into 2mL Type-I glass (Wheaton) vials and sealed with 13-mm rubber (Stelmi)stoppers. The solutions containing rHuPH20 were then split into twosets; one was kept as a refrigerated control until tested and the otherwas used for administration to the animals in this study. All dosingsolutions were kept refrigerated at all times and then returned fortesting. Each set of solutions were tested for rHuPH20 enzyme activityon the same date, within 1-6 days of being formulated.

Six adult male Yucatan pigs (S&S Farms, Ramona, Calif.), each weighingbetween 21 and 25 kg at the initiation of the study, were equipped withsurgically implanted jugular vein or carotid artery catheters withexterior vascular access ports installed for easy blood samplingthroughout the study. The animals were quarantined for 7 days prior toinstrumentation and treatment. Six animals were randomized to two studygroups as shown in Table 36, below. The animals were assigned to one oftwo groups each containing 3 animals per group and the assignment wasmaintained for cycles 1 and 2. For a third dosing cycle, two animalswere dropped due to non-patency of the cannulae, and the remaining fouranimals were reassigned with only 2 animals per group. Group 1 animal IDnumbers were 540, 541, and 542 for cycles 1 and 2; and 542 and 544 forcycle 3. Group 2 animal ID numbers: 544, 545, and 546 for cycles 1 and2; 545 and 546 for cycle 3.

The dosing solutions were administered subcutaneously (SC) into the leftflank of each pig behind the midline of the body. Prior toadministration of test article, a pre-treatment blood sample wasobtained. Animals received a single SC dose of the appropriate testarticle (0.2 U/kg; with animal measured prior to each administration toaccurately determine the correct dose) in an every-other day protocol.Each animal received a single SC bolus dose of the indicated insulin(i.e., either insulin or lispro) at 0.2 U/kg in either a vehicle or in afresh co-formulation of rHuPH20. After administration of the testarticle, at least 0.7-1.0 mL blood was serially withdrawn at 3, 6, 9,12, 15, 20, 25, 30, 45, 60, 90, 120, 180 and 240 minutes. Apre-treatment bleed (pre-bleed) also was taken prior to administration.The blood samples were immediately placed into serum tubes containing noanti-coagulant, placed on ice for a minimum of 30 minutes, thencentrifuged at 9500×g for 5 minutes at ambient temperature. The serumwas then transferred into a pre-labeled tube, frozen, and stored at −80°C., until all samples were shipped to Millipore for bioanalysis for theimmunoreactive insulin (IRI) levels.

TABLE 36 Dosing protocol for validation of pig model Dosing Study Group#1 Group #2 Cycle Day Day Treatment Treatment 1 1 0 Humalog ® insulinHumulin ® R insulin lispro 2 2 Humulin ® R Humalog ® insulininsulin/rHuPH20 lispro/rHuPH20 3 4 Humulin ® R insulin Humalog ® insulinlispro 4 6 Humalog ® insulin Humulin ® R lispro/rHuPH20 insulin/rHuPH202 5 8 Humalog ® insulin Humulin ® R insulin lispro 6 10 Humulin ® RHumalog ® insulin insulin/rHuPH20 lispro/rHuPH20 7 12 Humulin ® Rinsulin Humalog ® insulin lispro 8 14 Humalog ® insulin Humulin ® Rlispro/rHuPH20 insulin/rHuPH20 3 9 26 Humalog ® insulin Humulin ® Rinsulin lispro 10 28 Humulin ® R Humalog ® insulin insulin/rHuPH20lispro/rHuPH20 11 30 Humalog ® insulin Humulin ® R lispro/rHuPH20insulin/rHuPH20 12 32 Humulin ® R insulin Humalog ® insulin lispro

B. Serum Insulin Levels

The serum IRI concentrations were determined for each serum sample byinterpolation from a standard curve using StatLIA® assay analysissoftware (Brendan Technologies, Carlsbad, Calif.). Table 37 provides theIRI concentration following administration of Humalog® insulin lispro,Humalog® insulin lispro/rHuPH20, Humulin® R insulin or Humulin® Rinsulin/rHuPH20. Table 37 sets forth the baseline IRI levels, asmeasured in the pre-bleed samples. These baselines were then subtractedfrom the actual IRI concentrations measured at each timepoint todetermine the baseline-adjusted IRI concentration.

The mean serum IRI concentration-time profiles for each treatment (asseen, when plotted on a graph with IRI concentration on the Y axisversus time on the X axis) was similar over multiple cycles. In alldosing cycles, the pharmacokinetics of Humalog® insulin lispro andHumulin® R insulin were accelerated when co-administered subcutaneouslyin the rHuPH20 formulation. Any observed differences between treatmentswere substantially the same among treatment cycles, indicating theobserved differences were due to the treatment and were stable acrossthe cycles, over approximately 5 weeks of testing.

TABLE 37 Serum IRI concentration-time profiles Mean IRI concentration(pM) and standard deviation (SD) Humalog ® Humalog ® Humulin ® R insulininsulin lispro/ Humulin ® R insulin/ Time lispro rHuPH20 insulin rHuPH20(minutes) Mean SD Mean SD Mean SD Mean SD 0 (pre-bleed 37.0 52.3 45.037.5 20.9 27.3 19.2 23.8 baseline) Baseline-adjusted IRI concentrations0 0 0 0 0 0 0 0 3 8.1 25.2 65.5 79.3 0.7 3.0 113.0 109.7 6 20.6 46.1110.3 84.6 2.9 7.1 161.6 119.4 9 21.5 40.9 166.0 122.5 3.7 10.9 168.1136.2 12 40.6 71.6 143.0 99.0 7.3 20.7 166.0 118.0 15 52.3 78.4 262.2215.5 11.2 32.8 155.6 114.8 20 80.4 125.6 265.3 169.2 22.6 33.7 210.8172.4 25 93.6 92.5 263.3 190.0 40.7 48.2 253.0 171.9 30 119.7 116.7335.2 220.4 61.3 55.1 224.1 146.5 45 193.9 139.7 296.1 183.3 105.6 103.8253.1 188.1 60 164.0 106.8 206.5 150.3 107.0 91.4 172.4 115.3 90 115.276.4 101.8 74.4 100.4 95.4 137.0 110.0 120 95.7 68.9 72.3 63.6 105.557.6 93.9 63.4 180 24.6 23.6 38.5 45.4 105.5 81.7 50.5 49.7 240 15.221.7 65.8 106.5 81.8 126.3 33.8 50.9

C. Insulin Pharmacokinetics

The insulin concentration-time profile after subtraction of the baselineinsulin concentration (Table 37, above) was used to calculate thefollowing PK parameters: including t_(max), C_(max), Early t_(50%), Latet_(50%), and AUC_(interval). PK parameters were derived bynon-compartmental analysis using model 200 in WinNonlin Professionalversion 5.2 (Pharsight Corp., Mountain View, Calif.). Calculations ofstatistics were performed using SAS version 9.1.3 (SAS Institute, Cary,N.C.). All analyses were performed using a mixed model with fixedeffects for treatment. A compound symmetric covariance matrix amongrepeated observations for each animal was assumed. Analyses for C_(max)and all AUC endpoints were performed using log-transformed values withvalues of zero replaced by 1 prior to log transformation (zero on thelog scale). The time based endpoints were analyzed on the originallinear scale.

A summary of the pharmacokinetics of insulin following subcutaneousadministration of Humalog® insulin lispro or Humulin® R insulin,delivered alone (control) or with rHuPH20, is provided in Table 38. Thevarious PK parameters for each insulin delivered alone or with rHuPH20is shown as Mean±SD. The % control for each parameter (% controlcalculated by [mean (geometric or arithmetic) PK value for insulin withrHuPH20]/[mean (geometric or arithmetic) PK value for insulinalone]×100) also is provided in the table. The % control calculationswere based on Geometric Mean and p-value for log transformed data forC_(max) and AUC parameters, while based on arithmetic mean anduntransformed values for t_(max) and Early & Late t_(50%). N=16 pigsunless otherwise noted.

Table 39 sets forth a comparison of PK parameters of Humalog® insulinlispro alone to Humulin® R insulin with rHuPH20. The PK values areprovided as Mean±SD. Also provided is the % Humalog® insulin lispro(i.e. [mean (geometric or arithmetic) PK value for Humalog® insulinlispro with rHuPH20]/[mean (geometric or arithmetic) PK value forHumalog® insulin lispro alone]×100). The % control calculations werebased on Geometric Mean and p-value for log transformed data for C_(max)and AUC parameters, or based on arithmetic mean and untransformed valuesfor t_(max) and Early & Late t_(50%). N=16 unless otherwise noted.

TABLE 38 Insulin PK parameters following subcutaneous administrationwith or without rHuPH20 Humalog ® insulin lispro Humulin ® R insulinwith % with % alone rHuPH20 control P-value alone rHuPH20 controlP-value C_(max) 250 ± 140 417 ± 229 163 0.0251 214 ± 122 360 ± 180 1650.0218 (pmol/L) Early t_(50%)  36 ± 20 a  11 ± 6 a 32 0.0182 61 ± 49  10± 8 a 17 <0.0001 (min) t_(max) (min) 58 ± 26 39 ± 39 67 0.1963 94 ± 6138 ± 41 40 0.0004 Late t_(50%)  110 ± 42 a  52 ± 24 a 47 0.0002  170 ±49 b  70 ± 41 a 42 <0.0001 (min) AUC interval (min × nmol/L) 0-15 min0.35 ± 0.64 1.77 ± 1.24 1961 0.0004 0.06 ± 0.17 2.06 ± 1.28 43542<0.0001 0-30 min 1.65 ± 2.07 5.84 ± 3.59 763 0.0198 0.56 ± 0.73 5.33 ±3.30 1429 0.0027 0-1 hr 6.7 ± 4.8 14.2 ± 8.6  214 0.2776 3.4 ± 2.5 12.1± 7.3  246 0.2003 0-last 18.0 ± 8.8  26.6 ± 14.6 146 0.1195 21.3 ± 12.126.9 ± 16.0 116 0.5312 0-infinity  24.3 ± 8.6 c  32.2 ± 17.1 a 1170.5176  45.0 ± 41.2 c  32.6 ± 16.7 a 88 0.6118 1-4 hr 12.2 ± 6.8  12.9 ±7.8  106 0.8410 18.2 ± 10.3 15.0 ± 10.1 72 0.2259 2-4 hr 4.9 ± 3.2 6.0 ±4.9 215 0.3763 12.0 ± 7.8  6.8 ± 5.4 32 0.1930 a N = 15 b N = 4 c N = 13

TABLE 39 Insulin PK parameters following subcutaneous administration ofHumalog ® insulin lispro alone or Humulin ® R insulin with rHuPH20.Humulin ® R Humalog ® insulin with % Humalog ® insulin lispro rHuPH20insulin lispro P-value C_(max) (pmol/L)  250 ± 140  360 ± 180 143 0.0944Early t_(50%)   36 ± 20_(a)   10 ± 8_(a)29 0.0141 (min) t_(max) (min)  58 ± 26   38 ± 41 64 0.1631 Late t_(50%)  110 ± 42_(a)   70 ± 41_(a)64 0.0092 (min) AUC interval (min × nmol/L) 0-15 min 0.35 ± 0.64 2.06 ±1.28 2698 <0.0001 0-30 min 1.65 ± 2.07 5.33 ± 3.32 744 0.0212  0-1 hr 6.7 ± 4.8 12.1 ± 7.3 189 0.3646 0-last 18.0 ± 8.8 26.9 ± 16.0 1460.1196 0-infinity 24.3 ± 8.6_(b) 32.6 ± 16.6_(a) 128 0.3179  1-4 hr 12.2± 6.8 15.0 ± 10.1 121 0.4801  2-4 hr  4.9 ± 3.2  6.9 ± 5.4 290 0.2203_(a)N = 15 _(b)N = 13

D. Summary

Co-administration of Humulin® R insulin or Humalog® insulin lispro withrHuPH20 in pigs significantly altered specific PK parameters relative tocontrol injections (i.e. Humulin® R insulin or Humalog® insulin lisproalone). Specifically, the maximum exposure (C_(max)) was increased 163%for Humalog® insulin lispro (p=0.0251) and 165% for Humulin® R insulin(p=0.0218) when administered with rHuPH20 relative to the respectivecontrols. The onset of action (Early t_(50%)) was accelerated from 36 to11 minutes for Humalog® insulin lispro (p=0.0182) and from 61 to 10minutes for Humulin® R insulin (p<0.0001). The time of maximum effect(t_(max)) was accelerated from 58 to 39 minutes for Humalog® insulinlispro (p=0.1963) and from 94 to 38 minutes for Humulin® R insulin(p=0.0004). The Late t_(50%) was accelerated from 110 to 52 minutes forHumalog® insulin lispro (p=0.0002) and from 170 to 70 minutes forHumulin® R insulin (p<0.0001). Total exposure (AUC_(inf)) was notmeaningfully altered for either Humalog® insulin lispro (117% control;p=0.5176) or Humulin® R insulin (88% control; p=0.6118). Cumulativeexposure was shifted to earlier time windows for both Humalog® insulinlispro (AUC₀₋₃₀ increased 763% compared to when Humalog® insulin lisprowas administered alone; p=0.0198) and Humulin® R insulin (AUC₀₋₃₀increased 1429% compared to when Humulin® R insulin was administeredalone; p=0.0027). Coadministration of either Humulin® R insulin withrHuPH20 or Humalog® insulin lispro with rHuPH20 increased the absorptionrate of insulin to the vascular compartment (compared to when therespective insulin was delivered alone) as evidenced by a reduction intime to maximum serum IRI concentrations (t_(max), Early t_(50%), Latet_(50%)), and an increase in peak exposure concentrations (C_(max))compared to Humulin® R insulin or Humalog® insulin lispro alone. Inaddition, early cumulative exposure (AUC₀₋₃₀) was increased for bothHumalog® insulin lispro and Humulin® R insulin when coadministered withrHuPH20, compared to when administered alone.

The increase in peak exposure and acceleration of exposure uponadministration of either Humalog® insulin lispro and Humulin® R insulinwith hyaluronidase coadministration were observed broadly withoutmeaningful impact on animal, sequence, or cycle, and closely mirror theprevious human studies (see Example 1). Therefore, the pig is a suitablemodel for studying the effect of hyaluronidase on the absorption ofprandial insulin preparations.

Example 12 Pharmacokinetics of Regular Insulin at Two Doses Administeredwith and without rHuPH20 Subcutaneously

The pharmacokinetics (PK) of regular insulin, when subcutaneouslyadministered at two different concentrations, both alone andco-administered with rHuPH20, was assessed in the porcine modeldescribed in Example 10, above. A multiple dose 4-way crossover designstudy was conducted to compare the PK of regular insulin atconcentrations of 20 and 100 U/mL, when administered alone, to the sametwo concentrations after co-administration with rHuPH20. In each case, atotal of 0.2 U/kg of insulin was administered.

A. Dosing and Sampling

Four test articles were prepared for dosing. Two test articles contained20 U/mL and 100 U/mL regular insulin (Humulin® R insulin; Eli Lilly),respectively (designated Insulin U20 and Insulin U100, respectively).The remaining two test articles contained 20 U/mL and 100 U/mL regularinsulin (Diosynth Biotechnologies (a division of Schering-Plough),respectively, with 20 μg/mL (approximately 2400 U/mL) rHuPH20(designated Insulin-PH20 U20 and Insulin-PH20 U100, respectively). TheInsulin U20 test article was prepared by diluting Humulin® R insulin(100 U/mL; Lot A390566A; Eli Lilly) 1:5 with sterile diluent (EliLilly). The Insulin U100 test article was undiluted Humulin® R insulin(100 U/mL; Lot A509721; Eli Lilly). The Insulin-PH20 U20 test articlecontained 0.74 mg/mL (20 U/mL) regular insulin (Lot # SIHR107; DiosynthBiotechnologies) and 20 μg/mL (approximately 2400 U/mL) rHuPH20 in 25 mMTris, 120 mM NaCl, 0.01% Polysorbate 80, pH 7.3. The Insulin-PH20 U100test article contained 3.69 mg/mL (100 U/mL) regular insulin (Lot #SIHR107; Diosynth Biotechnologies) and 20 μg/mL (approximately 2400U/mL) rHuPH20 in 25 mM Tris, 120 mM NaCl, 0.01% Polysorbate 80, pH 7.3.

Six adult male Yucatan mini pigs (S&S Farms, Ramona, Calif.), eachweighing between 21 and 25 kg at the initiation of the study, had acatheter surgically implanted either in the jugular vein or the carotidartery to enable serial blood samples to be drawn over the duration ofthe study. The animals were randomized to two study groups, eachcontaining 3 ammonals/group as shown in Table 40. The group assignmentwas maintained for cycles 1 and 2. Each animal received two cycles oftreatment with all four test articles to facilitate comparison of thereproducibility of the insulin pharmacokinetics over a series of dosingcycles.

Test articles were administered subcutaneously (SC) into the left flankof each pig behind the midline of the body. Each animal received asingle SC bolus dose of the indicated insulin at 0.2 U/kg in an everyother day protocol. For the Insulin U20 and insulin-PH20 U20 testarticles, 10.0 μL/kg was administered. For the Insulin U100 andInsulin-PH20 U100 test articles, 2.0 μL/kg was administered. Bloodsamples (0.7-1.0 mL in volume) were collected prior to administration(pre-bleed), then at 3, 6, 9, 12, 15, 20, 25, 30, 45, 60, 90, 120, 180and 240 minutes post administration. The blood samples were placed intoserum tubes containing no anti-coagulant, placed on ice for a minimum of30 minutes, then centrifuged at 9500×g for 5 minutes at ambienttemperature. The serum was then transferred into a pre-labeled tube,frozen, and stored at −80° C. until samples were shipped to MilliporeBioPharma Services (St. Charles, Mo.) to determine the levels ofimmunoreactive insulin (IRI).

TABLE 40 Dosing protocol Cycle Dose Day Group 1 Group 2 1 1 0Insulin-PH20 U100 Insulin U20 2 2 Insulin-PH20 U20 Insulin U100 3 4Insulin U100 Insulin-PH20 U20 4 6 Insulin U20 Insulin-PH20 U100 2 5 8Insulin-PH20 U20 Insulin U100 6 10 Insulin U20 Insulin-PH20 U100 7 12Insulin-PH20 U100 Insulin U20 8 14 Insulin U100 Insulin-PH20 U20

B. Serum Insulin Levels

The serum IRI concentrations were determined for each serum sample byinterpolation from a standard curve using StatLIA® assay analysissoftware (Brendan Technologies, Carlsbad, Calif.). Table 41 provides themean serum IRI concentration following administration of Insulin U20,Insulin U100, Insulin-PH20 U20 and Insulin-PH20 U100. Table 41 setsforth the baseline IRI levels, as measured in the pre-bleed samples.These baselines were then subtracted from the actual IRI concentrationsmeasured at each timepoint to determine the baseline-adjusted IRIconcentration.

The insulin concentration-time profiles after each cycle of dosing werecompared for each treatment group. The mean serum IRI concentration-timeprofiles for each test article, as observed when plotted on a graph withIRI concentration on the Y axis versus time on the X axis) was similarover both cycles. In both dosing cycles, the PK of insulin wasaccelerated when co-administered subcutaneously with the rHuPH20formulation for both concentrations. Additional statistical models thatincluded fixed effects for treatment, sequence, cycle, thetreatment-by-cycle interaction, and animal within sequence for data fromcycles 1 and 2 were constructed for primary and secondary PK parameters(primary PK parameters included: Area Under the Curve (AUC) for assignedwindows of time, C_(max), t_(max), Early t_(50%), and Late t_(50%)%;secondary PK parameters included more detailed time windows for AUC, MRT(last and infinity), Lambda z, HL Lambda z, Clearance, and Volume ofDistribution), and showed that there is no systematic effect ofsequence, cycle, or animal, nor is there an interaction between cycleand treatment for the any of these variables.

TABLE 41 Serum IRI concentration-time profiles Mean IRI concentration(pM) and standard deviation (SD) Insulin-PH20 Insulin-PH20 Time InsulinU20 Insulin U100 U20 U100 (minutes) Mean SD Mean SD Mean SD Mean SD 0(pre-bleed 91.6 53.0 89.0 49.6 114.3 55.8 71.8 48.4 baseline)Baseline-adjusted IRI concentrations 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3139.7 472.2 63.1 73.4 2.2 5.6 49.4 41.3 6 74.2 234.6 178.7 269.3 8.117.6 88.8 73.7 9 206.3 594.2 122.0 101.3 12.9 30.9 144.5 85.0 12 93.0166.7 181.5 150.5 36.9 58.0 182.1 138.6 15 83.1 116.2 214.4 151.5 58.676.9 225.3 165.6 20 136.3 180.8 367.1 590.3 85.1 109.6 251.8 133.3 25139.1 147.7 247.3 161.1 174.5 262.2 285.7 142.8 30 238.0 292.2 294.1183.9 169.2 224.9 357.3 199.9 45 203.3 117.5 249.0 133.5 134.3 147.6234.7 115.8 60 185.5 127.6 174.8 101.8 102.5 65.1 252.1 169.1 90 106.167.7 131.2 128.1 85.4 87.8 222.7 144.7 120 70.8 71.9 73.0 47.0 70.9 61.7153.7 54.8 180 78.0 92.3 64.2 119.3 87.4 101.3 69.4 59.1 240 25.7 32.226.0 44.7 23.0 36.9 23.3 26.5

C. Insulin Pharmacokinetics

The insulin concentration-time profile after subtraction of the baselineinsulin concentration (Table 41, above) was used to calculate thefollowing PK parameters: including t_(max), C_(max), Early t_(50%), Latet_(50%), and AUC_(interval). Serum IRI versus time data were modeled bynon-compartmental analysis using WinNonlin Professional model 200(Version 5.2, Pharsight Corp., Mountain View, Calif.) and the PKparameters calculated. Calculations of statistics and statisticalcomparisons between groups were performed using SAS version 9.1.3 (SASInstitute, Cary, N.C.). All analyses were performed using a mixed modelwith fixed effects for treatment. A compound symmetric covariance matrixamong repeated observations for each animal was assumed. Analyses forC_(max) and all AUC endpoints were performed using log-transformedvalues with values of zero replaced by 1 prior to log transformation(zero on the log scale). The time based endpoints were analyzed on theoriginal linear scale.

A summary of the pharmacokinetics of insulin following subcutaneousadministration of Insulin U20, Insulin U100, Insulin-PH20 U20 andInsulin-PH20 U100, is provided in Table 42. The various PK parametersfor each insulin delivered alone or with rHuPH20 is shown as Mean±SD.The % control for each parameter (% control=[PK value for insulin withrHuPH20]/[PK value for insulin alone]×100) also is provided in thetable. The % control calculations were based on Geometric Mean andp-value for log transformed data for C_(max) and AUC parameters, while %control calculations were based on arithmetic mean and untransformedvalues for t_(max) and Early & Late t_(50%). N=16 pigs unless otherwisenoted.

TABLE 42 Insulin PK parameters following subcutaneous administrationwith or without rHuPH20 Insulin 20 U/mL Insulin 100 U/mL Insulin-Insulin- Insulin rHuPH20 % Insulin rHuPH20 % 20 U 20 U control P-value100 U 100 U control P-value C_(max) 429 ± 508 462 ± 567 106 0.8439 238 ±237 420 ± 208 237 0.0095 (pmol/L) Early t_(50%)  23 ± 14b 12 ± 5  520.1622  35 ± 38c 12 ± 7  34 0.0063 (min) t_(max) (min) 57 ± 42 39 ± 2168 0.2462 64 ± 49 47 ± 29 74 0.2775 Late t_(50%)  84 ± 38b 77 ± 45 920.7689 109 ± 64c 112 ± 53  103 0.9315 (min) AUC interval (min × nmol/L)0-15 min 1.61 ± 4.37 1.95 ± 1.73 1920 0.0045 0.27 ± 0.40 1.75 ± 1.027104 <0.0001 0-30 min  .79 ± 6.24 6.35 ± 5.90 623 0.0567 2.14 ± 2.835.89 ± 2.89 2400 0.0015 0-1 hr 10.02 ± 8.60  13.75 ± 8.92  179 0.20736.19 ± 6.78 13.98 ± 6.0  489 0.0012 0-last 24.4 ± 11.6 27.5 ± 20.5 1070.8777 18.5 ± 16.0 34.7 ± 14.6 354 0.0038 0-infinity  30.1 ± 10.3d 29.8± 22.1 89 0.7027  27.8 ± 19.1e  44.8 ± 13.2c 214 0.0161 1-4 hr 14.6 ±9.6  14.2 ± 12.2 97 0.9436 13.2 ± 11.3 22.2 ± 11.2 270 0.0204 2-4 hr 7.6± 6.5 6.5 ± 9.1 104 0.9720 8.1 ± 8.2 9.5 ± 5.4 398 0.1832 bN = 11animals cN = 10 animals dN = 8 animals eN = 9 animals

D. Summary

This study examined the effect of subcutaneously administering the sametotal insulin dose at different concentrations, with and withoutrHuPH20.

In the absence of co-administration with rHuPH20, reduction in theinsulin concentration from 100 U/mL to 20 U/mL resulted in fasterinsulin absorption with an increase in peak insulin concentration andgreater cumulative insulin exposure both early and, to a lesser extent,overall. Relative to control 100 U/mL injections, reducing theconcentration to 20 U/mL 1) increased C_(max) 91% from a geometric meanof 158 to 302 pmol/L; 2) reduced mean early t_(50%) from 35 to 23minutes, t_(max) from 64 to 57 minutes, and Late t_(50%) from 109 to 84minutes; and 3) increased geometric mean AUC₀₋₁₅ 300% from 20 to 80,AUC₀₋₃₀ 256% from 222 to 791, and AUC_(last) 131% from 9,021 to 20,820all in units of pmol×min/L.

Co-administration of regular insulin at either concentration withrHuPH20 also resulted in faster absorption following subcutaneousinjection relative to insulin alone. However, at the lower insulinconcentration of 20 U/mL, the relative increases over insulinadministered alone were not as dramatic as the insulin was alreadyabsorbed faster at 20 U/mL when delivered alone (as described above).

At the 100 U/mL concentration, which more typically is used by diabeticpatients, co-injection with rHuPH20 1) increased C_(max) 137% from ageometric mean of 158 to 375 pmol/L (p=0.0095); 2) reduced mean earlyt_(50%) from 35 to 12 minutes (p=0.0063), while t_(max) and Late T_(50%)were not significantly changed; and 3) increased geometric mean AUC₀₋₁₅70-fold from 20 to 1438 (p<0.0001), AUC₀₋₃₀ 23-fold from 222 to 5337(p=0.0015), and AUC_(last) 250% from 9,021 to 31,905 (p=0.0038) all inunits of pmol×min/L, compared to administration of insulin alone at the100 U/mL concentration.

At the lower insulin concentration of 20 U/mL, co-administration withrHuPH20 resulted in the following effects on insulin. PK, compared toadministration of insulin alone: 1) C_(max) was not significantlyaltered with geometric means of 302 and 322 pmol/L (p=0.84); 2) meanearly t_(50%) trended lower from 23 to 12 minutes (p=0.16), whilet_(max) and Late t_(50%) were not significantly changed; and 3)Geometric mean AUC₀₋₁₅ increase 18-fold from 80 to 1533 (p=0.0045),AUC₀₋₃₀ 5-fold from 791 to 4934 (p=0.0567), and AUC_(last) was unchangedat 20,820 and 22,184 (p=0.88) all in units of pmol×min/L.

The increase in peak exposure and acceleration of exposure uponadministration of rHuPH20 and regular insulin at 100 U/mL relative tocontrol insulin injection without rHuPH20, closely mirror the previoushuman studies (Example 1) pig study (Example 10). These results furtherdemonstrate that insulin kinetics also can be accelerated byadministration at a lower concentration, which is consistent with arate-limiting insulin hexamer dissociation step which is concentrationdependent (i.e. when insulin is administered subcutaneously alone, it isabsorbed when it dissociates from a hexamer to monomers, a process thatoccurs at lower concentrations of insulin). When co-administered withrHuPH20, this dependence on insulin concentration is greatly reduced oreven eliminated. Thus, the hyaluronidase dispersing effect ofco-administration of rHuPH20 with insulin can reduce the unwanted slowdown in insulin pharmacokinetics that is observed with injection ofinsulin at higher concentrations.

Example 13 Effect of Salt Concentration on rHuPH20 in the Presence ofMethylparaben

The effect of NaCl on the stability of rHuPH20 with and without thepreservative methylparaben, at accelerated temperature (40° C.) wasevaluated. Twelve different formulations were prepared by combiningrHuPH20 (10 mg/ml in histidine/HCl, pH 6.5, 130 mM NaCl) with sixdifferent concentrations of NaCl, with or without Methylparaben (Fluka).Each formulation contained 10 μg/mL rHuPH20, 25 mM Tris, pH 7.3, 0.01%Tween 80 and either 0, 50 mM, 100 mM, 150 mM, 200 mM or 250 mM NaCl withor without 0.2% methyparaben. The solutions were aliquotted into 2 mltype I glass vials with rubber stoppers and sealed with alumina capsduring the study. One set of vials was stored at 40° C. for four days,and the other set was kept in the refrigerator at 2-8° C. to serve as apositive control. The samples were then tested for hyaluronidase(enzymatic) activity. To evaluate the level of aggregates, sizeexclusion chromatography (SEC) was performed using a G2000 SWXL column(Tosoh Bioscience) the following conditions with 1×PBS as running bufferand a flow rate set at 1 ml/min.

Table 43 sets forth the results of the study, including hyaluronidase(enzymatic) activity, % main peak (i.e. the percentage of rHuPH20 thatwas contained in the main peak) and % aggregate peak (i.e. thepercentage of rHuPH20 that was contained in the peak attributed toaggregates). It was observed that the stability of rHuPH20 was sensitiveto the concentration of NaCl. In general, when the formulations wereincubated at 40° C., as the NaCl concentration decreased, the enzymaticactivity of rHuPH20 decreased. However, when stored in refrigerator at2-8° C., the rHuPH20 retained enzymatic activity regardless of theformulation. At elevated temperature, when NaCl was completelyeliminated from the solution, the entire activity of rHuPH20 was lost,whether there was methylparaben or not. The loss of enzymatic activitywas reduced as the NaCl concentration increased. There was significantdifference in enzymatic activity (paired t-test, P=0.0228) betweensamples with and without added methylparaben.

A similar correlation of NaCl concentration and the aggregate levels ofrHuPH20 was observed. The aggregate levels increased with decreasingNaCl concentration when samples were stored at elevated temperature.There were essentially no changes with or without added methylparabenwhen stored at 2-8° C. The formulations stored at −40° C. containingmethyparaben formed significantly more aggregate than those formulationsthat did not contain methylparaben (paired t-test, P=0.0058).

Thus, both the enzymatic activity and percent monomer of rHuPH20 asassessed by SEC were significantly reduced in formulations containingmethylparaben as compared to those formulations that contained nomethylparaben. Further, within the NaCl concentration range tested(0-250 mM), there was a direct relationship between NaCl concentrationand increased rHuPH20 stability.

TABLE 43 Enzymatic activities and SEC results of the samples stored 4days at 40° C. and 4° C. Enzymatic activity (U/mL) % main peak %aggregate peak 4° 40° 4° 40° 4° 40° Formulation C. C. C. C. C. C. NoNaCl, 12410 <LOD 99.65 0 0.35 100 0.2% MP 50 mM NaCl 12470 2990 99.222.86 0.78 97.14 0.2% MP 100 mM NaCl, 12380 3530 100 13.32 0 86.68 0.2%MP 150 mM NaCl, 13510 6200 100 26.31 0 73.69 0.2% MP 200 mM NaCl, 112506220 99.49 51.84 0.51 48.16 0.2% MP 250 mM NaCl, 10740 7340 100 65.55 034.45 0.2% MP No NaCl, 10430 <LOD 99.4 0 0.6 100 no MP 50 mM NaCl, 123703070 99.34 22.05 0.66 77.95 no MP 100 mM NaCl, 12580 9930 99.47 72.810.53 27.19 no MP 150 mM NaCl, 12750 11180 99.48 88.16 0.52 11.84 no MP200 mM NaCl, 13660 13340 99.64 96.22 0.36 3.78 no MP 250 mM NaCl, 1137011090 100 98.05 0 1.95 no MP LOD = limit of detection

Example 14 Co-Formulations of Insulin and rHuPH20

A series of studies were performed to assess the stability of rHuPH20and insulin under various conditions, such as various temperatures andpH, and formulations.

1. Effect of Osmolarity and pH on rHuPH20

In the first study, the effect of osmolarity and pH on the stability ofrHuPH20 (formulated as Hylenex recombinant (Hyaluronidase HumanInjection) was assessed by preparing formulations with varying saltconcentrations and pH values, and assessing any loss of activityfollowing storage under refrigerated (5° C.), accelerated (25° C.), andstress (25° C., 35° C. and 40° C.) conditions for up to 3 months.Hylenex recombinant (Hyaluronidase Human Injection) contains 150 U/mLrHuPH20, 144 mM NaCl, 10 mM Sodium phosphate dibasic, 1 mg/mL humanalbumin human, 2.7 mM Edetate disodium, 2.7 mM CaCl, and has anosmolality range of 290 to 350 mOsm and a pH of 7.4. This formulationwas adjusted to prepare the 8 formulations (and control Hylenex) setforth in Table 44. The enzymatic activity (i.e. hyaluronidase activitywas determined as described above. rHuPH20 content also was determinedby RP-HPLC.

No meaningful changes were observed at the recommended (5° C.) oraccelerated (25° C. or 30° C.) storage conditions for the four solutionsprepared at the pH and osmolality specification limits or the controlsolution at recommended storage conditions. rHuPH20 was observed to bestable at pH 7.4 and generally more stable under acidic rather thanbasic conditions, as assessed by loss of enzyme activity and loss ofrHuPH20 content. The effect of ionic strength was more modest. Atelevated temperatures, formulations containing higher ionic strengthappeared to be slightly more stable than those with lower ionicstrength. There was a significant decrease in stability between 35° C.and 40° C.

TABLE 44 Formulations of rHuPH20 Formulation (adjustment NaCl Osmolaritymade) mM mg/mL % Hylenex (mOsm/kg) pH Plus 21% 120 7.0 83 267 7.5 volumeH₂0 Plus 10% 132 7.7 91 290 7.5 volume H₂0 Plus 19 mM 164 9.6 100 3507.4 NaCl Plus 91 mM 236 13.8 98 450 7.3 NaCl Control 145 8.5 100 325 7.4Plus 5 mM 150 8.8 100 328 6.5 HCl Plus 8 mM 153 8.9 99 331 5.5 HCl Plus1.9 mM 145 8.5 100 324 8.6 NaOH Plus 2.2 mM 145 8.5 100 323 9.5 NaOH

2. Effect of pH on rHuPH20

The effect of varying the pH of the buffer system on the stability ofrHuPH20 was assessed. rHuPH20 (1,200,000 U/mL, 10 mg/mL) was formulatedin 130 mM NaCl, 10 mM histidine, with a pH of 5.0, 5.5, 6.0, 6.5 or 7.0.The formulations were then stored at 5° C. for 0, 3, 6, 9 and 12 months;25° C. in 60% relative humidity for 0, 3, 6, 9 and 12 months, and 35° C.for 0, 1, 2, 3 and 6 months. At refrigerated temperatures, allformulations were stable over all time periods. The rHuPH20 remainedwithin trend limits for 12 months at 5° C., 6 months at 25° C., and 3months at 35° C. for the test articles at pH 6.0, 6.5, and 7.0. Theformulations prepared at pH 5.0 and 5.5 were more sensitive to elevatedtemperature, resulting in a significant decrease in enzymatic activity.

3. Effect of pH and Preservative on rHuPH20 Formulated with InsulinAnalogs

To evaluate the impact of pH and preservative on rHuPH20 stabilityformulated with insulin analogs at refrigerated (5° C.), accelerated(30° C. and 35° C.), and agitation (25° C.) storage conditions for up to4 weeks, rHuPH20 was combined with Humalog® insulin lispro or Novolog®insulin aspart and the enzymatic activity and stability assessed. Theinsulin stability was assessed by RP-HPLC. The test articles wereprepared with 10 μg/mL rHuPH20, 100 U/mL insulin analog, 140 mM NaCl, 20mM Tris HCl with either 0.2% phenol; 0.2% m-Cresol; 0.2% paraben; 0.2%phenol and 0.1% F68; or 0.2% phenol and 1 mM benzoate. Each of theseformulations was prepared at pH 7, 7.25 and 7.5, resulting in a total of30 test articles (15 Humalog® insulin lispro/rHuPH20 and 15 Novolog®insulin aspart/rHuPH20 test articles). The test articles were thenstored at 5° C., 30° C., 35° C. and 25° C. with agitation for 4 weeks.The rHuPH20 enzymatic activity was assessed under all conditions.Insulin solubility was assessed by RP-HPLC for test articles stored at5° C., and 25° C. with agitation.

It was observed that rHuPH20 activity was not affected by eitherpreservative or pH after 4 weeks at 5° C. Under agitation stressconditions (20° C.), the activity of rHuPH20 was not affected whenco-formulated with Novolog® insulin aspart and any of the preservativesat any the tested pH. In contrast, in some formulations with Humalog®insulin lispro, such as when formulated with 0.02% phenol, m-cresol orphenol/benzoate, the activity of rHuPH20 after 6 hours was reduced by upto 75%, most typically as the pH increased. This loss of activitycorrelated with precipitation of Humalog® insulin lispro.

Table 45 sets forth the rHuPH20 activity retained in each of the testarticles after incubation at 30° C. and 35° C. A slight loss of rHuPH20activity to an average of about 85% of the original activity wasobserved at 30° C. A greater loss was observed at 35° C., particularly,for example, in test articles containing 0.2% m-Cresol or 0.2% parabenas the pH increased.

Novolog® insulin aspart remained stable and soluble in all formulationsunder all storage conditions. Although the solubility of Humalog®insulin lispro was retained at pH 7.5 after 4 weeks at 5° C., Humalog®insulin lispro precipitated at lower pH (7.0 and 7.25) at histemperature. Precipitation also was observed under agitation stressconditions after 6 hours.

TABLE 45 rHuPH20 activity remaining after 4 weeks at 30° C. and 35° C.in insulin analog/rHuPH20 formulations rHuPH20 activity remaining (%)30° C. 35° C. Humalog ® Novolog ® Humalog ® Novolog ® Form- insulininsulin insulin insulin ulation pH lispro aspart lispro aspart Phenol7.0 92 92 77 74 7.25 89 92 71 69 7.5 91 92 69 60 m- 7.0 82 78 40 29Cresol 7.25 85 81 29 21 7.5 77 71 11 9 Paraben 7.0 90 89 29 34 7.25 9089 20 22 7.5 81 79 8 10 Phenol/ 7.0 93 94 81 68 F68 7.25 91 92 75 61 7.590 73 63 13 Phenol/ 7.0 90 88 73 67 ben- 7.25 91 87 71 63 zoate 7.5 8886 64 58

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A method for controlling post-prandial glucose levels in a subject,comprising administering to the subject a fast-acting insulin analog anda hyaluronan-degrading enzyme less than 15 minutes before a meal upuntil 30 minutes after commencing a meal, wherein: the amounthyaluronidase administered is sufficient to render the fast-actinginsulin analog a super fast acting insulin; a super fast-acting insulinhas an onset of action that is faster than the analog alone, and aduration of action that is shorter than the analog alone, whereby, uponadministration, the super fast-acting insulin closely mimics thephysiologic post-prandial insulin response of a non-diabetic subject toachieve post-prandial glycemic control; the sufficient amount of ahyaluronan degrading enzyme to render the a super fast-acting insulincomposition is 1 Unit or more hyaluronidase activity/mL; andadministration of the fast-acting insulin analog and the hyaluronidasedegrading enzyme is effected via subcutaneous, intradermal,intramuscular or intraperitoneal administration.
 2. The method of claim1, wherein the fast-acting insulin analog and hyaluronidase degradingenzyme are administered at or at about the time of ingestion of a mealis commenced.
 3. The method of claim 1, wherein the fast-acting insulinanalog and hyaluronidase degrading enzyme are administered within 10minutes of before a meal.
 4. The method of claim 1, wherein thefast-acting insulin analog and hyaluronidase degrading enzyme areadministered within 5 minutes before a meal.
 5. The method of claim 1,wherein the fast-acting insulin and hyaluronan degrading enzyme areco-formulated to form a super fast-acting insulin composition.
 6. Themethod of claim 1, wherein the fast-acting insulin and hyaluronandegrading enzyme are administered separately.
 7. The method of claim 6,wherein the fast-acting insulin analog and hyaluronan degrading enzymeare administered sequentially or simultaneously.
 8. The method of claim1, wherein the fast-acting insulin analog and hyaluronan degradingenzyme are administered to the subject via syringe, insulin pen, insulinpump or a closed loop system.
 9. The method of claim 8, wherein thefast-acting insulin analog and hyaluronan degrading enzyme areco-formulated.
 10. The method of claim 8, wherein the fast-actinginsulin analog and hyaluronan degrading enzyme are administeredseparately.
 11. The method of claim 10, wherein the fast-acting insulinanalog and hyaluronan degrading enzyme are in separate reservoirs in aclosed loop system.
 12. The method of claim 1, wherein the dosage of thefast-acting insulin analog is less than the dosage of the fast-actinginsulin administered by the same route in the absence of the hyaluronandegrading enzyme.
 13. The method of claim 1, wherein the amount offast-acting insulin analog is from or from about 10 U/mL to at or about500 U/ml insulin; and the sufficient amount of a hyaluronan degradingenzyme to render the fast-acting insulin analog super fast-actinginsulin is functionally equivalent to at least or about hyaluronidaseactivity/mL, 2 U/mL, 3 U/mL, 4 U/mL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9U/mL, 10 U/mL, 15 U/mL, 20 U/mL, 25 U/mL, 30 U/mL, 35 U/mL, 37.5 U/mL,40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, 100 U/mL, 200U/mL, 300 U/mL, 400 U/mL, 500 U/mL, 600 U/mL, 700 U/mL, 800 U/mL, 900U/mL, 1000 U/mL, 2000 U/mL, 3000 U/mL or 5000 U/mL hyaluronidaseactivity/mL.
 14. The method of claim 1, wherein the sufficient amount ofa hyaluronan degrading enzyme to render the composition a superfast-acting insulin composition is functionally equivalent to at leastor about 30 to 35 Units hyaluronidase activity/mL.
 15. The method ofclaim 1, wherein the amount of insulin for a single dosage is or isabout 0.05 Units, 0.06 Units, 0.07 Units, 0.08 Units, 0.09 Units, 0.1Units, 0.2 Units, 0.3 Units, 0.4 Units, 0.5 Units, 0.6 Units, 0.7 Units,0.8 Units, 0.9 Units, 1 Unit, 2 Units, 5 Units, 10 Units, 15 Units, 20Units, 25 Units, 30 Units, 35 Units, 40 Units, 50 Units or 100 units;and/or the amount of hyaluronan degrading enzyme for a single dosage isfunctionally equivalent to at least or to about 0.3 Units, 0.5 Units, 1Unit, 2 Units, 5 Units, 10 Units, 20 Units, 30 Units, 40 Units, 50Units, 100 Units, 150 Units, 200 Units, 250 Units, 300 Units, 350 Units,400 Units, 450 Units, 500 Units, 600 Units, 700 Units, 800 Units, 900Units, 1000 Units, 2,000 Units, 3,000 Units, 4,000 Units or more ofhyaluronidase activity.
 16. The method of claim 1, wherein thefast-acting analog is insulin aspart, insulin lispro or insulinglulisine.
 17. The method of claim 16, wherein the insulin analog isselected from among an insulin with an A chain with a sequence of aminoacids set forth in SEQ ID NO:103 and a B chain with a sequence of aminoacids set forth in any of SEQ ID NOS:147-149.
 18. The method of claim 1,wherein the hyaluronan degrading enzyme is a hyaluronidase.
 19. Themethod of claim 18, wherein the hyaluronidase is a solublehyaluronidase.
 20. The method of claim 19, wherein the solublehyaluronidase is a PH20, or a truncated form thereof.
 21. The method ofclaim 20, wherein the PH20 is an ovine, bovine or truncated human PH20.22. The method of claim 21, wherein the hyaluronidase is a truncatedhuman PH20 that is selected from among polypeptides having a sequence ofamino acids set forth in any of SEQ ID NOS:4-9, or allelic variants orother variants thereof that have 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to a polypeptide set forthin any of SEQ ID NOS: 4-9, and that retain hyaluronidase activity. 23.The method of claim 22, wherein the hyaluronidase is a solublehyaluronidase provided as a composition designated rHuPH20.